Polyamine analogues as therapeutic and diagnostic agents

ABSTRACT

Novel inhibitors of polyamine transport having inhibition constants two orders of magnitude lower than those of known compounds are disclosed. These polyamine analogues are useful pharmaceutical agents for treating diseases where it is desired to inhibit polyamine transport or other polyamine binding proteins, for example cancer and post-angioplasty injury. Novel chemical synthetic methods to obtain polyamine analogues are disclosed, including the production of a combinational polyamine library. These approaches yield analogues with desirable activities both for diagnostic and research assays and therapy. The assays of the invention are useful for high throughput screening of targets in the discovery of drugs that interact with the polyamine system.

This application claims priority under 35 USC 119(e) over provisionalapplication Ser. Nos. 60/052,586, filed Jul. 15, 1997, 60/065,728, filedNov. 14, 1997 and 60/085,538, filed May 15, 1998 and this application isa 371 of PCT/US98/14896, filed Jul. 15, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the field of chemistry and biochemistry relates to thesynthesis and use of novel polyamine transport (PAT) inhibitor compoundswith pharmacological or agricultural uses and as probes for biochemicalassays or for purification of selected polyamine binding targets. Asdrugs, these compounds are used to treat disorders of undesired cellproliferation, primarily cancer, alone or combined with other agentssuch as polyamine synthesis inhibitors. An assay employing some of thesecompounds is useful for monitoring polyamine uptake or transport (PAT)and allows analysis of binding sites for polyamines or for other basicligands on a variety of molecules. This invention also relates to thesynthesis and use of novel polyamine combinatorial libraries. Theselibraries are used to discover compositions that inhibit PAT and/or thatbind to a cellular polyamine transporter (PATr). Various members ofthese libraries or compounds discovered through use of the librarieshave utility as drugs, agricultural chemicals, and as probes. Thisinvention also identifies key elements that comprise the polyaminebinding sites of membrane as well as soluble proteins.

2. Description of the Background Art

Polyamines are ubiquitous molecules that provide a “buffer” system forthe cell by modulating the activities of proteins, RNA, DNA, and lipids.Polyamines may play a direct role in apoptosis. Mammals and otherorganisms have an active polyamine uptake and recycling system thatcomplements their polyamine synthetic capabilities. Because polyaminesmodulate such a large range of molecules and cellular activities,polyamine analogues, as disclosed herein, offer novel approaches fortargeting a variety of disease states, particularly cancer, and alsoprovide unique tools to monitor cellular activities.

Polyamines and Cancer

The potential of polyamines as anticancer agents has been longrecognized. Polyamines affect chromatin structure in eukaryotes andprokaryotes by binding specifically to DNA (Balasundaram, D. et al.,Mol. Cell. Biol. 100:129-140, 1991) so that condensation occurs when thebinding sites on DNA are saturated. Acetylation of polyamines andhistones lowers their affinity for DNA and is believed to occur intandem to alter the structure and function of the nucleosome, thusregulating DNA replication and transcription by loosening DNA at theends of the core particle. Because polyamines are absolutely essentialfor DNA replication, they are of interest in the treatment of cancer.Particular interest has been focused on preventing cell proliferation bylowering intracellular polyamine levels. Polyamine analogues asdescribed herein are useful for preventing or treating cancer and otherproliferative diseases by acting at a number of different levels. Thepresent invention focuses on the inhibition of PAT. Other targetsinclude induction of spermine/spermidine acetyltransferase (SSAT),hypusine modification, and other proteins that inhibit the cell cycle orinduce apoptosis.

The Polyamine Transporter (PATr)

The increased demand for polyamines by rapidly growing, transformedcancer cells is only partially met by an increased rate of synthesis. Toexploit this increased need for polyamines, synthesis inhibitors havebeen sought. Additionally, lowering polyamine concentrations can resultin aberrations in chromatin structure leading to cell death orinhibition of proliferation (Quemener, V. et al., Anticancer Res.14:443-448, 1994; Porter, C. W. et al., Cancer Res. 53:581-586, 1993).It has become increasingly apparent that the initial disappointingresults observed in the clinic with polyamine synthesis inhibitorsarises from compensatory increases in transport of polyamines by aspecific active transport system (Seiler, N. et al., Int. J. Biochem.22:211-218, 1990; Seiler, N. et al., J. Biochem. Cell. Biol. 28:843-861,1996). The promising results observed in cell culture with a suicidesubstrate inhibitor of ornithine decarboxylase,α-difluoromethylornithine (DFMO), or with an inhibitor ofS-adenosylmethionine decarboxylase, methylglyoxal bis(guanylhydrazone)(MGBG) did not transfer to human clinical trials (Schecter, P. J. etal., In Inhibition of Polyamine Metabolism. Biological Significance andBasis for New Therapies; McCann, P. P. et al., eds; 1987, pp 345-364).Since the only two avenues for carbon transfer into polyamine pools aresynthesis or transport, simultaneous inhibition of both of thesepathways is considered by the present inventors to be a promisinganti-cancer therapeutic approach.

A study confirming the validity of this chemotherapeutic approach usedtransplanted murine L1210 leukemia cells that were deficient in PAT.Mice transplanted with the wild-type L1210 cancer cells (with intactPAT) died after 12 days, even when treated with DFMO. In contrast, DFMOmice transplanted with PAT-deficient L1210 cells lived longer than 60days (Ask, A. et al., Cancer Lett. 66:29-34, 1992). These authors alsoshowed that treatment of mice harboring wild-type L1210 cells with acombination of (1) DFMO (2) a low polyamine diet and (3) antibiotics(which decrease polyamine production by gut flora) resulted in prolongedsurvival compared to treatment with DFMO alone.

Augmented PAT into cancer cells promotes cell killing. J. L. Holley etal. (Cancer Res. 52:4190-4195, 1992) showed up to a 225-fold increase incytotoxicity of a chlorambucil-spermidine conjugate compared tochlorambucil alone. A series of nitroimidazole-polyamine conjugates werealso effective (Holley, J. L. et al., Biochem. Pharmacol. 43:763-769,1992). Others showed that mice infected with a multi-drug resistantstrain of malaria were cured by treatment with achloroquinoline-putrescine conjugate (Singh, S. et al., J. Biol. Chem.272:13506-13511, 1997). Thus, the effectiveness of cytotoxic compoundscould be enhanced by their conjugation with polyamines. These effectsmay have been due to the exploitation of the PAT system to deliver thesecompounds into cancer cells. The present invention is therefore directedin part to rapid and efficient testing of many different conjugatesbetween polyamines and known drugs for their transport into cells.Furthermore, as described below, this invention combines the cytotoxicproperties of known drugs with the facilitated transport of polyamines,which relies on the present inventors' discoveries surrounding the PATrdescribed herein. By accessing the database ofstructure-activity-relationships (SARs) of PATr substrates, the presentinventors are able to predict the transportability of a novel chemicalentity or a novel polyamine conjugate.

Polyamine Transport (PAT) Assays

There is no known high-throughput assay for measuring PAT. Aradiochemical assay is used for biochemical analysis of transport andhas been used to study PAT in yeast and a variety of mammalian cells(Kakinuma, Y. et al., Biochem. Biophys. Res. Comm. 216:985-992, 1995;Seiler, N. et al., Int. J. Biochem. Cell Biol. 28:843-861, 1996). See,for example Huber, M. et al. Cancer Res. 55:934-943, 1995.

The radiometric assay uses radiolabeled polyamines such as putrescine,spermidine or spermine, but, due to the low signal, large numbers ofadherent or non-adherent cells are required. Additional care is requiredwith spermine due to its non-specific adsorption to cells and plastics.Cells are mixed with the test compounds and the radiolabeled polyamineto initiate the assay. The cells are incubated for 1-60 minutes,depending on cell type. The assay is terminated by removal of the mediumand cooling the plates to 4° C. The cells are then washed with coldmedium three times, dissolved in 0.1% sodium dodecyl sulfate and theradioactivity in solution is then determined by scintillation counting.This assay is difficult to scale up to a high throughput procedure dueto the low signal from the radiolabel and the handling requirementsinherent in procedures with radioactivity.

Combinatorial Approaches to Polyamines and Analogues

Combinatorial chemistry, a rapidly changing field of molecularexploration, is still in its infancy. For reviews, see Lam, K. S.,Anticancer Drug Des. 12:145-167, 1997; Salemme, F. R. et al.; Structure5:319-324, 1997; Gordon, E. M. et al., J. Med. Chem. 37:1385-1401, 1994;Gallop, M. A. et al., J. Med. Chem. 37:1233-1251, 1994). Thepharmaceutical industry, is now realizing that the original approach ofthe combined synthesis of hundreds to thousands of compounds in one“flask” followed by testing and deconvoluting the results is a tediousprocess with many pitfalls. The more traditional approach of medicinalchemistry, that is, the synthesis and testing of one compound at a time,yields more reliable and informative results about thestructure-activity relationship (SAR) around a target. The trend incombinatorial chemistry is therefore towards synthesis of multiplecompounds at once, with each in a separate container. Therefore, manyhave adopted this one-compound/one-well parallel synthetic approach tomolecular exploration. While many lead compounds have been generatedthis way, the chemistries do not necessarily lead to a molecule with thenecessary drug-like characteristics.

Combinatorial chemistry, a rapidly changing field of molecularexploration, is still in its infancy. For reviews, see Lam, K. S.,Anticancer Drug Des. 12:145-167, 1997; Salemme, F. R. et al.; Structure5:319-324, 1997; Gordon, E. M. et al., J. Med. Chem. 37:1385-1401, 1994;Gallop, M. A. et al., J. Med. Chem. 37:1233-1251, 1994). Thepharmaceutical industry, is now realizing that the original approach ofthe combined synthesis of hundreds to thousands of compounds in one“flask” followed by testing and deconvoluting the results is a tediousprocess with many pitfalls. The more traditional approach of medicinalchemistry, that is, the synthesis and testing of one compound at a time,yields more reliable and informative results about the SAR around atarget. The trend in combinatorial chemistry is therefore towardsynthesis of multiple compounds at once, with each in a separatecontainer. Therefore, many have adopted this one-compound/one-wellparallel synthetic approach. While many lead compounds have beengenerated this way, the chemistries do not necessarily lead to amolecule with the necessary drug-like characteristics.

Polyamine analogues are notoriously difficult to synthesize. Due to thepolycationic nature of the final products, traditional chromatographictechniques such as silica gel chromatography cannot be used.Intermediates need a lipophilic protecting group that enablespurification of the compounds and extraction with organic solvents.Bergeron has solved some of these problems through the use of themesityl-type amino protecting group (Bergeron, R. J. et al., J. Med.Chem. 40:1475-1494, 1997), which not only solved the problems ofhandling (allowing purification by silica gel and extraction by organicsolvents), but also gave a synthetic handle to extend the backbone ofthe polyamine. After treatment with NaH, a sodium amide anion isproduced which can be alkylated with an alkyl halide to extend thebackbone.

Although this approach extends synthetic possibilities somewhat, it isstill significantly limited. Use of the mesityl group requires that aharsh reagent like HBr/HOAc be employed for removal, thereby limitingthe substituents of the resulting polyamine acid-stable ones. Theavailability of suitable alkyl halides, together with the amino startingmaterials, is also limited. Therefore, while this approach has madesignificant inroads toward simpler analogue production, it is severelylimited in its potential for structural diversity. Other syntheticapproaches suffer from similar limitations (Moya, E. et al., InNeuropharmacology of Polyamines; Carter, C., ed.; Academic Press:London, 1997; pp. 167-184). Several initial reports of solid phasesynthesis of polyamine analogues (Byk, G. et al., Tetrahed. Lett.38:3219-3222, 1997; Furka, A., Int. J. Peptide Protein Res. 37:487,1991) have serious limitations including a covalently attached linkerresidue and the lack of sufficient diversity of structural components.These deficiencies are effectively addressed by the present invention.

Induction of Spermine/Spermidine Acetyltransferase (SSAT)

Cellular levels of polyamines are tightly regulated so that only a smallwindow of variability in concentration is tolerated. This regulation ismediated by the control of polyamine synthesis, uptake and catabolism.Abnormally high concentrations of polyamines induce the enzyme SSATwhich is associated with apoptosis (Parchment, R. E. et al., Cancer Res.49:6680-6686, 1989). Polyamine analogues induce apoptosis by inductionof this enzyme (Ha, et al., Proc. Natl. Acad. Sci. 94:11557-11562, 1997;Albanese, L. et al., Biochem. J. 291:131-137, 1993) in a celltype-specific way, presumably due to the accumulation of the polyamineanalogue in the cell and its binding to a polyamine sensitive repressoror activator of SSAT transcription. Acetylated polyamines, the productsof the SSAT-catalyzed reaction, are substrates for the enzyme polyamineoxidase which generates stoichiometric release of H₂O₂ believed to beresponsible a more proximate cause of the apoptotic response.

Hypusine

The protein eIF-5A appears to play a role in protein synthesis, althoughits exact function remains obscure (Hanauske-Abelm, H. M. et al., FEBSLett. 266:92-98, 1995). EIF-5A is unique in that it is modified by theunusual amino acid hypusine. Hypusine is generated post-translationallyby the sequential action of deoxyhypusyl synthase (using spermidine as asubstrate) and deoxyhypusyl hydroxylase. Inhibition of this modificationof eIF-5A coincides with proliferative arrest late in the G1 phase ofthe cell cycle. This modification occurs in most, if not all,eukaryotes. The present inventors have noted that inhibitors ofdeoxyhypusyl synthase would be useful in treating diseases associatedwith unwanted cell proliferation, such as cancer, by blocking the cellcycle.

Inhibition of Angiogenesis

Inhibition of polyamine synthesis decreases the vascularization of solidtumors. One month of treatment with DFMO resulted in a 50% reduction inneoplastic vessel count in humans with cervical interepithelialneoplasia (Mitchell, M. F. et al., Proceedings AACR 39:Ab. 600, 1998).DFMO inhibited the neovascularization induced by tumor cells in vivo(Jasnis, M. A. et al., Cancer Lett 79:39-43, 1994). Squalene, apolyamine analogue, also inhibits angiogenesis in the rabbit corneaassay.

Other Mechanisms that Block Cell Growth or Induce Apoptosis

Transport, SSAT, and deoxyhypusine synthase are targets for developingtherapies for cancer and other proliferative diseases. The potentialpolyamine related targets associated with cancer have not beenexhausted. CHENSpm is polyamine analogue that induces apoptosis but doesnot function through any of the mechanisms described above (Ha, H. C.Proc Nat. Acad. Sci USA 94:11557-11562 (1997) CHENSpm does not induceSSAT, but does reduce spermidine and spermine levels and produces a G₂cell cycle arrest at subtoxic concentrations, suggesting an unusual modeof action. Polyamines are known to bind to tubulin and promote itsbundling, though this is just one of several possible mechanisms bywhich polyamines can induce apoptosis or inhibit cell growth. Forexample, some microtubule associated proteins (MAPs) also bindpolyamines.

Monitoring of Cancer-Related Molecules by Polyamine Analogues

Several polyamine binding anti-cancer targets could be monitored usingvarious polyamine analogues. Acetylated polyamines, the products of thespermine/spermidine acetyltransferase (SSAT) enzymatic reaction, aresubstrates for the enzyme polyamine oxidase. Oxidation of acetylatedpolyamines produces a stoichiometric release of H₂O₂ which is believedto be responsible for the apoptotic response. This induction is celltype-specific and is believed to be due to the accumulation of thepolyamine analogue in the cell and its possible binding to apolyamine-sensitive repressor or activator of transcription of SSAT.This repressor has not been identified, but a probe/assay for itsdetection would enable the synthesis of better drugs.

Membrane-bound Proteins

Several cellular receptors have polyamine binding sites that influencereceptor binding activity. Hypertension, osteoporosis, Alzheimer'sdisease and ischemia may all be targeted through polyamine bindingreceptors such as calcium receptor, N-methyl-D-aspartate (NMDA)receptors, glutamate receptors, Ca²⁺ channels and several of theinwardly rectifying K⁺ channels (Ventura, C. et al., Am. J. Physiol. 267H587-H592, 1994).

Polyamines in Other Diseases

Post-Angioplasty Injury

Because PAT inhibitors can contribute to inhibition of cell growth, theyare viewed by the present inventors as being useful in the treatment ofpost-angioplasty injury. Endothelial denudation and vessel wall injurylead to neointimal hyperplasia and luminal stenosis. Inhibition ofsmooth muscle cell proliferation, for example, could inhibit neointimalformation. According to this invention, this initiation of cellproliferation after injury is amenable to treatment with PAT inhibitorspreferably in combination with polyamine synthesis inhibitors (Takagi,M. M. et al., Arterioscler. Thromb. Vasc. Biol. 17:3611-3619, 1997;Nakaoka, T. et al., J. Clin. Invest. 100:2824-2832, 1997; Maillard, L.et al., Cardiovasc. Res. 35:536-546, 1997).

Hypertension

Ca²⁺ channels, which have high affinity binding sites for polyamines,are modulated by polyamine levels. Polyamines modulate theβ-adrenergic-mediated changes in Ca²⁺ levels and contractility (Ventura,C. et al., Am. J. Physiol. 267: H587-H592, 1994). Ca²⁺ channels andbinding can be measured as described for the NMDA receptor and theK⁺-inward rectifying channels in Ventura, supra. Thus, an appropriatepolyamine or analogue can be harnessed to modulate Ca²⁺ in place of thechannel blockers currently in use.

Osteoporosis

Blood and tissue (e.g., nerve) calcium levels are modulated by theexternal Ca²⁺ sensing receptor (CaR) located in the parathyroid andkidney. The CaR has a specific polyamine binding site. Modulation ofthis receptor is believed to be a promising approach to the treatment ofosteoporosis.

Alzheimer's Disease

The CaR plays a different role in the brain from that in theparathyroid. Aggregated β-amyloid protein in this disease can stimulatethe CaR and eventually lead to its down-regulation. Polyamines,likewise, can bind to the CaR and inhibit CaR down-regulation stimulatedby β-amyloid. Polyamines or polyamine analogues can therefore serve asprotective molecules.

Immunosuppression

Low dose methotrexate is a common treatment for rheumatoid arthritis(RA). The reason for its efficacy is unknown, although it is notbelieved to inhibit proliferation of lymphoid cells.S-adenosylmethionine (AdoMet) metabolism has been proposed to play adirect role in its immunosuppressive activity.

The direct effect of AdoMet metabolism on the immune response is notknown, though a role for polyamines has been suggested (Furumitsu, Y. etal., J. Rheumatology, 20:1661-1665, 1993; Nesher, G. et al., Arthr.Rheumat. 33:954-957, 1990). Cytokines are believed to play a direct rolein the pathogenesis of RA, and IL-2 is low in patients' synovial fluid,a condition which was reversed by inhibitors of polyamines (Flesher, E.et al., J. Clin. Invest. 83:1356-1362, 1987). The polyamine synthesisinhibitor DFMO prolongs the life of MRL-lpr/lpr mice, a model ofsystemic lupus erythematosus.

Polyamine levels are elevated in the urine, synovial fluid, synovialtissue, and peripheral blood mononuclear cells of RA patients. Culturingthese cells in the presence of methotrexate inhibited the production ofIgM-rheumatoid factor. Spermidine reversed this effect, indicating tothe present inventors that a combination of polyamine synthesisinhibitors and PAT inhibitors are useful can treat autoimmune diseases.Other polyamine analogues, spergualin and deoxyspergualin, areimmunosuppressive and may be beneficial for treating multiple sclerosis(Bergeron et al., J. Org. Chem. 52:1700, 1987; Drug Fut. 16:1165, 1991).

Psychiatric Disorders

A number of the compounds which the present inventors have found toinhibit PAT with high affinity, as disclosed herein, structurallyresemble several known antipsychotic or antidepressant drugs.

DNA/RNA-Polyamine Hybrids for Stable Binding to Nucleic Acids

The spacing between ammonium polycations in naturally occurringpolyamines (spermidine and spermine) is 3-4 carbons. This is the exactspatial separation for optimal binding to a DNA or RNA polyanionicphosphate backbone. It has been suggested that this interaction,together with the interaction with chromatin proteins, modulates genetranscription and expression. Recently, such ionic interactions havebeen exploited by combining polycationic 3′, 5′-polyguanidine linkersbound with the base portion of the nucleosides to enhance double ortriple helix formation. An oligomeric polyadenosyl RNA analogue in whichthe phosphodiester backbone units were replaced by cationic guanidineunits was inactive, since triple strand formation was decreased (Dempey,R. D. et al., Proc. Natl. Acad. Sci. U.S.A. 93:4326-4330, 1996; Goodnow,Jr., R. A. et al., Tetrahedron Lett. 38:3195-3198 and 3199-3202, 1997).These nucleic acid analogues melt like double helices consistent withWatson-Crick base pairing.

In this regard, this invention provides compositions and methods thatincorporate complex structural substituents onto a polyamine chain tooptimize the targeting of DNA or RNA for inhibiting replication,transcription or translation.

Other Polyamine-Binding Receptors as Targets

Of particular pharmacological interest are the ways in which polyaminesmodulate various receptor or channel functions. In particular,polyamines modulate the Ca⁺-permeable glutamate receptors assembled fromsubunits containing a glycine residue at the RNA editing site. Theinward rectification of the K⁺ inward rectifying channels is induced byblocking the outward current using cytoplasmic Mg²⁺ (or intrinsicchannel gating). This gating is due primarily to a block by cytoplasmicpolyamines (Shyng-Si, et al. Proc. Natl. Acad. Sci. USA. 93:12014-12019,1996). According to this invention, polyamine analogues are useful formodulating glutamate receptors that are important in ischemia, strokes,and cardiovascular disease. NMDA receptor antagonists act asanticonvulsants so that agents active at NMDA receptors are additionaluseful targets.

Anti-Infective Effects of Polyamine Modulation

Parasitic organisms such as Trypanososma cruzi are thought to obtain thepolyamines necessary for their growth from their hosts rather thansynthesize their own. DFMO (an ODC inhibitor), decreases theavailability of putrescine, a precursor of spermidine and sperminesynthesis. DFMO can cure T. brucei infection in mice and is activeagainst African sleeping sickness in humans caused by T. bruceigambiense. DFMO also has clinical utility in Pneumocystis cariniipneumonia and in infection by the coccidian protozoan parasite,Cryptosporidium. In the laboratory, DFMO acts against Acanthamoeba,Leishmania, Giardia, Plasmodia and Eimeria (Marton, L. J. et al., Annu.Rev. Pharmacol. Toxicol. 35:55-91, 1995). Polyamines are also essentialfor the growth of Hemophilus and Neisseria organisms (Cohen, S. S., AGuide to the Polyamines, Oxford University Press, NY. pp 94-121, 1998).Thus, compounds and methods of the present invention can be used totreat diseases caused by Trypanososma cruzi, T. brucei, Pneumocystiscarinii, Cryptosporidium, Acanthamoeba, Leishmania, Giardia, Plasmodia,Eimeria, Hemophilus and Neisseria.

Plant Pathogens

Lowering of polyamine levels may protect plants against a wide range offungi, e.g., Uremyces phaseoli Linnaeus, race O. Unifoliolate (beanrust). DFMO is an effective fungicide in the following plants: tomatoplants against Verticillium wilt fungus; wheat against stem rust fungusand powdery mild fungus; bean plants against powdery mildew fungus;Macintosh apple leaves against the powdery mildew fungus; Ogle oatsagainst leaf rust fungus; and corn against the corn rust fungus (U.S.Pat. No. 4,818,770). The compositions of this invention that lowerpolyamine levels by PAT inhibition or that have other actions on systemsthat utilize, or are affected by, polyamines could be useful inprotecting plants against a wide range of fungi.

Miscellaneous Targets

Polyamines may protect DNA from radiation damage (Newton, G. L. et al.,Radiat. Res. 145:776-780, 1996). Therefore, an agent that raisespolyamine levels, or that substitutes for an endogenous polyamine moreeffectively, may be a useful adjunct to radiotherapy. Polyamines mayplay an important role in controlling mammalian fertility (U.S. Pat. No.4,309,442) and maintaining embryonic growth (U.S. Pat. No. 4,309,442).Other actions ascribed to polyamines include anti-diarrheal,anti-peristaltic, gastrointestinal anti-spasmodic, anti-viral,anti-retroviral, anti-psoriatic and insecticidal (U.S. Pat. No.5,656,671).

The present invention also has use in: (a) cleanup of toxic orradioactive metal waste that requires specific ion-binding (this can bedesigned into a polyamine for use in vivo or environmentally); (b)image-enhancement in medical imaging systems such as X-ray,computer-assisted tomography or magnetic resonance technologies; (c)enzyme-like catalysis that is required in asymmetric organic synthesisor resolution; (d) xenobiotic detoxification; and (e) nucleosidaseactivities.

Combination Therapy

Certain agents inhibit growth of tumor cells in culture in a manner thatis additive with cytotoxic drugs. The agent 8-chloro-cAMP (8-Cl-cAMP)(Tortora et al., Cancer Res. 57:5107-5111, 1997) is a cAMP analogue thatselectively down-regulates PLA-1, a signaling protein that is directlyinvolved in cell proliferation and neoplastic transformation and thatmediates the mitogenic effects of certain oncogenes and growth factors.In nude mice bearing human GEO colon cancer xenografts, 8-Cl-cAMPinhibited tumor angiogenesis and secretion of growth factors of the EGFfamily and synergized with anti-EGF receptor antibodies in inhibitingtumor growth. 8-Cl-cAMP acts by different mechanisms than do thepolyamine analogues of this invention.

The PAT inhibitors of the present invention can be used alone, incombination with 8-Cl-cAMP, or in combination with an ODC inhibitorand/or a SAM decarboxylation inhibitor (with or without 8-Cl-cAMP).Because polyamine modulation affects chromatin structure, other agentscan be used in combination with the PAT inhibitors of this inventioninclude topoisomerase inhibitors, DNA alkylating agents and DNAintercalating agents such as doxorubicin, adriamycin, chlorozotocin,etc.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

The present invention is directed to a series of polyamine analogues orderivatives and their use as drugs, as agricultural or asenvironmentally useful agents. The invention defines sites andstructures within these compounds that are key to their binding (andpolyamine binding) to membrane (and soluble proteins), particularly thePATr.

The compositions of the present invention include polyamine derivativessubstituted at one or more positions. Disubstituted polyamines arepreferably substituted at the two terminal nitrogens, but may bealternatively or additionally substituted at internal nitrogen and/orinternal carbon atoms.

A preferred embodiment is a highly specific PAT inhibitor withpharmaceutical utility as an anti-cancer chemotherapeutic. Preferredcompounds with such activity include N¹-dansylspernine (also termedmonodansylspermine or MDS (1), N¹-dansylspermidine (also termedmonodansylspermidine or MDSd, N¹-[(N⁶-dansyl)-6-aminocaproyl]spermine(termed DACS, 4), N¹-[(N⁶-dansyl)-6-aminocaproyl]spermidine (DACSd),N¹-[(N⁶-5-(4-chlorobenzamidomethyl)-thiophene-2-sulfonyl)-6-aminocaproyl]spermine5 or N¹-[(N⁶-(2-dibenzofuransulfonyl)-6-aminocaproyl]spermine 6. Thelatter two compounds have surprisingly high binding and inhibitoryactivity compared to the corresponding compounds lacking the C6 caproylspacer between the aryl group and the polyamine. For this reason, DACS 4and DACSd, and compounds 5 and 6 are preferred pharmaceuticalcompositions. Use of alternate spacers (or linkers or couplers) andother aryl or heterocyclic “head” groups, all of which are disclosedherein, is expected to yield even more potent PAT inhibitors.

Preferred substituents are structures that increase binding affinity orotherwise enhance the irreversibility of binding of the compound to apolyamine binding molecule, such as the PATr, an enzyme or DNA. Suchadditional substituents include the aziridine group and various otheraliphatic, aromatic, mixed aliphatic-aromatic, or heterocyclicmulti-ring structures. Reactive moieties which, like aziridine, bindirreversibly to a PATr or another polyamine binding molecule, are alsowithin the scope of this invention. Examples of reactive groups thatreact with nucleophiles to form covalent bonds include chloro-, bromo-and iodoacetamides, sulfonylfluorides, esters, nitrogen mustards, etc.Such reactive moieties are used for affinity labeling in a diagnostic orresearch context, and subserve pharmacological activity as sites withina drug that inhibit PAT or polyamine synthesis. The reactive group canbe a reactive photoaffinity group such as an azido or benzophenonegroup. Chemical agents for photoaffinity labeling are well-known in theart (Flemming, S. A., Tetrahedron 51:12479-12520, 1995). Photoreactivecompounds for cancer treatment are also known in the art.

Specifically, a composition which is a polyamine analogue or derivativethat binds to a polyamine-binding site of a molecule and/or inhibitspolyamine transport, which composition has the formula

R₁—X—R₂

wherein

R₁ is H, or is a head group selected from the group consisting of astraight or branched C₁₋₁₀ aliphatic, alicyclic, single or multiringaromatic, single or multiring aryl substituted aliphatic,aliphatic-substituted single or multiring aromatic, a single ormultiring heterocyclic, a single or multiring heterocyclic-substitutedaliphatic and an aliphatic-substituted aromatic;

R₂ is a polyamine; and

X is CO, NHCO, NHCS, or SO₂

In another embodiment of the above composition, R₂ has the formula

NH(CH₂)_(n)NH(CH₂)_(p)NH(CH₂)_(q)NHR₃

wherein

(a) n, p and q vary independently and n=p=q=1 to 12;

(b) R₃ is H; C₁₋₁₀ alkyl; C₁₋₁₀ alkenyl; C₁₋₁₀ alkynyl; alicyclic; aryl;aryl-substituted alkyl, alkenyl or alkynyl; alkyl-, alkenyl-, oralkynyl-substituted aryl; gauanidino; heterocyclic;heterocyclic-substituted alkyl, alkenyl or alkynyl; and alkyl-,alkenyl-, or alkynyl-substituted heterocyclic.

The above composition may further comprise, linked between X and R₂, alinker L and an additional group y, such that said composition has theformula:

R₁—X—L—Y—R₂

wherein,

L is a C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, alicyclic, orheterocyclic;

X is CO, SO₂, NHCO or NHCS; and

Y is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.

In the foregoing compositions R₁ can have the formula:

wherein

R₄, R₅, R₆, R₇ and R₈ are, independently, H, OH, halogen, NO₂, NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH₂)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ wheren=0 to 10;

Alternatively, R₁ has the formula:

wherein

R₄ and R₅ are, independently, H, OH, halogen, NO₂, NH₂, NH(CH)_(n)CH₃,N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH₂)_(n)CH₃,NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃, where n=0 to 10;

In yet another embodiment, R₁ has the formula:

wherein

r and s vary independently and r=s=0 to 6;

R₄, R₅, R₆, R₇, R₈ and R₉ are, independently, H, OH, halogen, NO₂, NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH₂)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ wheren=0 to 10; and

Q is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.

Furthermore, R₁ may have the formula:

wherein

r and s vary independently and are 0 to 6;

R₄, R₅, R₆ and R₇ are, independently, H, OH, NO₂, NH₂, NH(CH)_(n)CH₃,N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH₂)_(n)CH₃,NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ where n=0 to 10; and

Q is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.

In the foregoing compositions, R₁ may be selected from the groupconsisting of naphthalene, phenanthrene, anthracene, pyrene,dibenzofuran, acridine, 2,1,3-benzothiodiazole, quinoline, isoquinoline,benzofuran, indole, carbazole, fluorene, 1,3-benzodiazine, phenazine,phenoxazine, phenothiazine, adamantane, camphor, pipiridine,alkylpiperazine, morpholine, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, thiophene, furan, pyrrole,alkyl-1,2-diazole, alkylimidazole, alkyl-1H-1,2,3-triazol,alkyl-1H1,2,3,4-tetrazole, thiazole, oxazole, 1,3,4-thiadiazole,pyridinyl, pyrimidine, 1,2-diazine, 1,4-diazine and 1,3,5-triazine,4-dimethylaminoazobenzene, 3-phenyl-5-methylisooxazole,3-(2-chlorophenyl)-5-methylisooxazole,2-(4-chloropheny)-6-methyl-7-chloroquinoline,6-chloroimidazo[2,1-β]thiazole, α-methylcinnamic acid, and2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole.

R₁ may also be a D- or L-amino acid.

Also provided is the above composition where R₁ has a formula selectedfrom the group consisting of

₁₂—R₁₃—Y₁—R₁₄  (A)

R₁₂Y₁R₁₃Z₁R₁₄  (B)

wherein

R₁₂ and R₁₃, independently, are H, naphthalene, phenanthrene,anthracene, pyrene, dibenzofuran, acridine, 2,1,3-benzothiodiazole,quinoline, isoquinoline, benzofuran, indole, carbazole, fluorene,1,3-benzodiazine, phenazine, phenoxazine, phenothiazine, adamantane,camphor, pipiridine, alkylpiperazine, morpholine, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, thiophene,furan, pyrrole, alkyl-1,2-diazole, alkylimidazole,alkyl-1H-1,2,3-triazol, alkyl-1H1,2,3,4-tetrazole, thiazole, oxazole,1,3,4-thiadiazole, pyridinyl, pyrimidine, 1,2-diazine, 1,4-diazine and1,3,5-triazine, 4-dimethylaminoazobenzene, 3-phenyl-5-methylisooxazole,3-(2-chlorophenyl)-5-methylisooxazole,2-(4-chloropheny)-6-methyl-7-chloroquinoline,6-chloroimidazo[2,1-β]thiazole, α-methylcinnamic acid, or2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole; and further,

wherein a ring of R₁₂, R₁₃ or both in formulas (A), (B) and (D), isoptionally substituted with one or more of OH, halogen, NO₂, NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH₂)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or COO(CH)_(n)CH₃,

where n=0 to 10;

R₁₄ and R₁₅, and, in formula (C), R₁₃, independently, are (CH₂)_(n),(CH₂)_(n)CH═CH, (CH₂)_(n)(CH═CH)_(m)CO, or (CH₂)_(n)CO where n=O to 5and

m=1 to 3;

Y₁ and Z₁, independently, are CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂,NHSO₂, SO₂—NHSO₂, SO₂, O, S, COO or

when R₁ is of formula (A) or (B), Y₁ represents a bond between a C or Natom of R₁₂ and a C or N atom of R₁₃ and Z₁ represents a bond between aC or N atom of R₁₃ and a C or N atom of R₁₄; or

when R₁ is of formula (C) or Y₁ represents a bond between the C and a Cor N atom of R₁₃ and Z₁ represents a bond between the C and a C or Natom of R₁₄; or

when R₁ is of formula (D) Y₁ represents a bond between a C or N atom ofR₁₂ and a C or N atom of R₁₄ and Z₁ represents a bond between a C or Natom of R₁₃ and a C or N atom of R₁₅.

In the above compositions, R₂ preferably has the formula

NHCH(Z₁)(CH₂)_(n)NH(CH₂)_(p)NH(CH₂)_(q)CH(Z₁)NHR₃

wherein

(a) n, p and q vary independently and n=p=q=1 to 12;

(b) R₃ is H; C₁₋₁₀ alkyl; C₁₋₁₀ alkenyl; C₁₋₁₀ alkynyl; alicyclic; aryl;aryl-substituted alkyl, alkenyl or alkynyl; alkyl-, alkenyl-, oralkynyl-substituted aryl; gauanidino or heterocyclic; and

(c) Z₁ is CH₃, CH₂CH₃ or cyclopropyl.

In another embodiment, R₂ has the formula:

wherein

x=1 to 4; y=1 to 3,

R₁₀ and R₁₁ are, independently, H, (CH₂)_(n)NHR₁₂ or(CH₂)_(k)NH(CH₂)₁NHR₁₂ where n=k=1=1 to 10, and R₁₂ is H or C(N=H)NH₂

In the above compositions, R₂ is preferably selected from the groupconsisting of N¹-acetylspermine, N¹-acetylspermidine,N⁸-acetylspermidine, N¹-guanidinospermine, cadaverine,aminopropylcadaverine, homospermidine, caldine (horspermidine),7-hydroxyspermidine, thermine (norspermine), thermospermine,canavalmine, aminopropylhomospermidine, N,N′-bis(3-aminoppropyl)cadaverine, aminopentylnorspermidine,N⁴-aminopropylnorspermidine, N⁴-aminopropylspermidine, caldopentamine,homocaldopentamine, N⁴-bis(aminopropyl)norspermidine, thermopentamine,N⁴-bis(aminopropyl)spermidine, caldohexamine, homothermohexamine,homocaldohexamine, N-(3-aminopropyl)-1,3-propanediamine,N,N′-bis(3-aminopropyl)ethylendiamine,N,N′-bis(3-aminopropyl)-1,4-piperazine,N,N′-bis(3-aminopropyl)-1,3-piperazine,N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-bis(2-aminoethyl)-1,3-propanediamine, tris(3-aminopropyl)amine, andtris(aminoethyl)amine

Preferred compositions are polyamine analogues selected from the groupconsisting of compounds designated herein 3, 4, 5, 6, 13, 14, 29, 40,43, 44, 45, 57, 58, 56, 66, 67, 72, 76, 84, 88, 89, 95 and 96, mostpreferably, compound 4, 5, 6, 43, 65, 66, 84, 89, 95 or 96.

R₁ or R₃ may be bonded at one or more sites to a reactive moiety that iscapable of forming covalent bonds with a nucleophilic site on a targetmolecule, such as a protein or a nucleic acid, preferably a cellularreceptor or other cell surface molecule. Such composition permitessentially irreversible binding that is advantageous in both diagnosticand therapeutic uses.

The present invention is also directed to a pharmaceutical compositionuseful for treating a disease or condition in which the inhibition ofpolyamine transport is desirable, comprising a composition as describedabove and a pharmaceutically acceptable excipient. The pharmaceuticalcomposition may further include a an inhibitor of polyamine synthesis;preferably DFMO. Other combinations include the above pharmaceuticalcomposition and one or more additional agents known to be useful fortreating said disease or condition

This invention also provides a method for treating a disease or acondition in a subject associated with undesired cell proliferationand/or which is treatable by inhibition of polyamine transport,comprising administering to said subject an effective amount of apharmaceutical composition as described above. The undesired cellproliferation may be associated with proliferation of cells of theimmune system, cell of the vascular neontima, tumor cells or withundesired angiogenesis. Preferred diseases to be treated as aboveinclude cancer or post-angioplasty injury.

The present invention is directed to a series of polyamine analoguesuseful in an improved assay of polyamine uptake into the cell orpolyamine binding to specific ligands. The invention identifies elementsthat are key for polyamine binding to membrane proteins such as the PATr(PATr), and to soluble-proteins, and which can be monitored through thistechnique.

Disubstituted polyamines, preferably having a reactive group at one end,may also be employed as assay or biochemical probes.

A preferred assay method employs a monosubstituted polyamine probehaving a moiety that serves as a detectable label (a “reporter”),preferably a fluorophore, most preferably the dansyl group, or anothersubstituent that can be detected through a variety of means, includingby ELISA. A preferred assay method employs a polyamine or analogueimmobilized to a solid support.

Additional substituents which may be present on the polyamine core (withor without the reporter group), are structures which increase bindingaffinity, or otherwise enhance the irreversibility of binding of thecompound to a polyamine binding molecule, such as a PATr, an enzyme orDNA. Such additional substituents include the aziridine group andvarious other aliphatic, aromatic or heterocyclic multi-ring structures.A reactive moiety, which, like aziridine, can bind irreversibly to aPATr or another polyamine binding molecule is also contemplated.Examples of groups which react with nucleophiles to form covalent bondsinclude chloro-, bromo- and iodoacetamides, sulfonylfluorides, esters,nitrogen mustards, etc. Such reactive moieties are used for affinitylabeling in a diagnostic or research context, and subservepharmacological activity as parts of drugs that inhibit PAT or polyaminesynthesis. The reactive group can also be a reactive photoaffinity groupsuch as an azido- and benzophenone group. Chemical reagents inphotoaffinity labeling are well-known (Flemming, S. A., Tetrahedron51:12479-12520, 1995). Moreover, photoreactive compounds for cancertreatment are known in the art.

The present invention includes a high throughput screening assay whichallows processing of high numbers of potential target compounds that arebeing screened for activity as inhibitors, preferably competitiveinhibitors, of PAT. The method is suited to the 96 well microplateformat for use with robotic sample and plate handling systems known inthe art. The means for detecting transport are a function of thedetectable label used. Fluorescence and chemiluminescence are themethods of choice. The present invention also encompasses a uniquepharmacophore for a polyamine binding site that can be used to isolate apolyamine binding target or to assay the conformational state of aselected target by its binding.

Also provided is an enzymatic assay which permits amplification of thesignal, wherein biotin or some other “reporter” is conjugated to thepolyamine as the “detectable label,” and is detected by allowing thebinding of streptavidin conjugated to an enzyme, followed by generationof a colored product of the enzyme from a chromogenic substrate. Alsoincluded is a composition in which both biotin and a “hapten” grouprecognized by an antibody are coupled to a polyamine. In anothercomposition the hapten is coupled directly to the enzyme. This compoundcan be captured by an antibody specific for the hapten and detected bythe biotin interacting with streptavidin-enzyme complex as above.Alternatively, streptavidin can be used for capture and the antibody fordetection. In either case, signal amplification permits a significantincrease in sensitivity.

Specifically, the diagnostic or assay composition is designed to be usedin an assay of polyamine transport or polyamine binding, and comprises apolyaminc analogue as set forth above, that is detectably labeled and/orincludes at least one reporter group capable of detection. Preferablythe analogue comprises a linker group L between the reporter group andsaid polyamine.

As indicated above, a preferred reporter group is a fluorophore, achromophore or a luminescer, most preferably dansyl or biotinyl; thepolyamine is preferably spermine, spermidine or putrescine. Mostpreferred for this utilityis monodansylspermine or DACS.

The diagnostic composition may also include as a single, or as one ofseveral reporter groups, a hapten recognized by an antibody.

The above diagnostic compositions may have the polyamine analogue boundto an enzyme.

In preferred embodiment, the polyamine analogue is immoblized to a solidsupport.

An assay method provided herein for detecting polyamine transportcomprises

(a) incubating cells with a composition as above; and (b) detecting thepresence of said reporter group in said cells. When applied in ascreening assay to identify unknown compounds for their binding to apolyamine binding site or their entry into a cell via a polyaminetransporter, the assay comprises (a) incubating molecules or cellshaving a polyamine binding site or cells having a polyamine transporterwith (i) a polyamine analogue of any of claims 26-33, (ii) with andwithout said unknown compound; (b) measuring the quantity of saidreporter bound to said cells or molecules or internalized in said cells;and (c) comparing the amount of reporter bound or internalized in thepresence of said unknown compound to the amount of reporter bound orinternalized in the absence of said unknown compound, wherein areduction in the amount of said reporter detected is measure of thebinding or transport of said unknown compound.

The methods of the present invention include high throughput solid phasesynthesis of a polyamine library. This library includes the attributesof a solid phase support, a cleavable linker that attaches the moleculeto the support, the addition of extenders that are a series of protectedaldehydes, amino acids, etc., that can be coupled and subsequentlyreduced to amines through reductive amidation, and a variety ofterminator molecules. This combination allows for the synthesis of alarge variety of novel analogues that can be used for many of thetargets and assays described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure and activity relationships (SAR) betweenspermidine, MDS and DACS. K_(i) values are the inhibitory constantsobtained in a PAT inhibition assay.

FIG. 2 (sheets 2/1 to 2/10) is a tabular representation of a largenumber of chemical structures 3-98 that were tested for their effects oncell growth. R, an index of growth inhibitory activity, is the ratio ofthe growth of cells in the presence of the test compound to the growthin the presence of the compound plus DFMO. The K_(i), (inhibitionconstant) reflects a compound's inhibition of PAT in cell culture. Thesebiological effects provide a basis for SAR analysis.

FIG. 3 shows synthetic routes to N¹-substituted polyamine analogues99-102.

FIG. 4 is a scheme of the synthesis ofN-(1-anthracenyl)-N′-(N¹-spermidyl)urea 9

FIG. 5 is a scheme of the synthesis of N¹-(1-pyrenylsulfonyl)spermine 15

FIG. 6 shows a scheme of the synthesis ofN¹-((1-carbonyl)-4-(1-pyrenyl)butane)spermine 7

FIG. 7 shows a scheme of the synthesis of the synthesis ofN¹-dansyl-spermine 3 (MDS).

FIGS. 8 and 9 each show a different synthetic scheme for the synthesisof DACS.

FIG. 10 shows four classes (111-114) of conformationally restrictedpolyamine analogues, and at the bottom, a stereochemically defined,internally cyclic polyamine analogues (116).

FIG. 11 is a synthetic scheme wherein free primary amino groups areblocked by N-acylation (44) and N-alkylation (77), thereby reducingpotential metabolic degradation of the derivatized PAT inhibitors.

FIG. 12 is a synthetic scheme for bis α-gem-dimethylpolyamine analogues121.

FIG. 13 is a synthetic scheme for internally substituted polyamineanalogues containing cyclopropyl groups (122-126)

FIG. 14 is a synthetic scheme for internally substituted polyamineanalogues containing a C—C branch (127-134)

FIG. 15 shows examples of spacers or linkers for use with multiring headgroup (135-139).

FIG. 16 shows a series of compounds (140-147) containing multiple ringhead groups.

FIG. 17 is a graph showing the effects of DACS on growth of MDA breastcancer cells with and without DFMO.

FIG. 18 is a graph showing the effects of headless polyamine analogueson growth of PC-3 prostate cancer cells with and without DFMO.

FIG. 19 lists chiral carbon-substituted amino acid linker groups.

FIG. 20 is a scheme of the synthesis ofN¹-(aziridinyl)-N¹²-[(N⁶-dansyl)-6-aminocaproyl]spermine 157.

FIG. 21 is a scheme of the synthesis of a di-substituted aziridinylpolyamine analogue 160.

FIG. 22 is a graph showing inhibition of the growth of MDA-MB-231 cells,by DACS in the presence (▪) or absence (♦) of the polyamine synthesisinhibitor DFMO. See also, FIGS. 2/1-2/10 for the effects of a largenumber of polyamine analogues on PAT and tumor cell growth. Cells wereplated in the presence of varying concentration of DACS with and without1 mM DFMO. Cells numbers (expressed as % of controls) were determinedafter 6 days as above.

FIG. 23 is graph showing inhibition of cell growth in the presence of 1μM spermidine.

FIG. 24 is a graph showing the inhibition of growth of PC-3 prostatecancer cells by the combination of DACS and DFMO. See description ofFIG. 22 for conditions and details.

FIG. 25 shows a group of chemical structures (161-165) including threeknown psychoactive compounds trifluoperazine 163, thorazine 164 andimipramine 165. Compounds 161, 162 and 165 inhibited polyaminetransport.

FIG. 26 is a graph showing the inhibition of spermidine/spermineacetyltransferase (SSAT) enzymatic activity by DACS.

FIG. 27 is a graph showing a comparison of the kinetics of uptake ofN′-monodansyl spermine (MDS) with the uptake of radiolabeled spermidine.MDS concentrations were as follows: ♦0 ▴1 μM ▪0.3 μM 3 μM

FIG. 28 is a graph showing detection of MDS in the absence of DFMO byfluorescence in A172 glioblastoma cells.

FIGS. 29 and 30 describe the synthesis of a biotin modified polyaminesN¹[(N⁶-(biotinyl)-6-aminocaproyl)]spermine and N¹-(biotinyl)spermine.

FIG. 31 is a schematic illustration showing the possible sites formodifying a polyamine to create an “immobilization handle” and a“reporter handle” combination.

FIG. 32 is a graph showing the detection of N1-dansylspermine and DACSusing the enzymatic detection system

FIG. 33 is a general scheme that brings together the three majorcomponents of the present compositions in a synthetic cycle forgenerating polyamine derivatives.

FIG. 34 outlines synthesis of an activated tert-alkoxycarbonyl MeO-PEGpolymer which is reacted with a free amino/protected aldehyde extendersynthon.

FIG. 35 shows the production of these extenders from either commerciallyavailable amino alcohols or the chiral amino acid precursor pool.

FIG. 36 shows the next step in the synthetic cycle: reductive aminationwith NaBH₃CN is used to initially extend the backbone followed by anadditional reductive amination step with an aldehyde to terminate thesecondary amine produced.

FIG. 37 shows the final steps, including the final capping and theacid-mediated cleavage of the product from the polymeric support as thetrifluoroacetate salt of the desired analogue.

FIG. 38 shows “modifications” of polyamine analogues as they areextended with aldehydic nucleoside terminators. Each amino group can bedressed individually and specifically with any of the fourribonucleosides or 2′-deoxyribonucleosides.

FIG. 39 shows an example of a solid support with alternative linkinggroups used for solid phase synthesis of polyamine libraries.3,4-dihydro-2H-pyran-2-yl-methoxymethyl polystyrene is shown.

FIG. 40 shows various linkers used in a multipin method of dimensionallystable polypropylene/polyethylene pins to which a graft polymer iscovalently linked. The Rink amide linker is shown as structure 23acoupled to the pin.

FIG. 41 shows a compound that is synthesized using a solid support andthe synthetic approach described for FIGS. 4 and 5. Compound 31 a issynthesized using the blocked 3-aminopropanal 27a as the first extender,benzaldehyde 28a as the first terminator, the blocked methioninal 29a asthe second extender and acetone as the final terminator.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have designed novel compounds for therapeutic usesand have devised tests using such compounds as probes for measuring PATand polyamine binding in an efficient, high throughput assay. Using thenovel methods, they have screened for and discovered compounds with highaffinity for the PATr that inhibit uptake, both competitively andnon-competitively. Such compounds are useful as drugs in a number ofdiseases, particularly cancer. They can also be used as a component ofnovel drug combinations with, for example, a polyamine synthesisinhibitor such as DFMO (which inhibits ornithine decarboxylase) or withother agents. The compounds of the present invention are also useful inother diseases or conditions in which polyamines play a role asdescribed above, and have agricultural and environmental uses.

The inventors found that various chemical groups can be attached to apolyamine to give it advantageous properties as an inhibitor of PAT oras a probe in an assay of PAT and for drug screening. Such chemicalmodification does not destroy the effective binding and, in fact,enhances the affinity of the derivatized polyamine for the PATr. Hence,these compounds are useful for discovery of inhibitors of polyamineuptake.

Definitions

As used herein, the term “polyamine” is intended to mean putrescine,spermine or spermidine, as well as longer linear polyamines, branchedpolyamines, and the like, which may have between 2 and about 10nitrogens. Also included in this definition are polyamine derivatives oranalogues comprising a basic polyamine chain with any of a number offunctional groups bound to a C atom or a terminal or internal N atom. Apolyamine derivative may include a terminal linker or spacer groupbetween the polyamine core and a derivatizing function.

A “head group” is defined as a moiety bonded either directly to thepolyamine or attached to a linker that is bonded to the polyamine. It ispreferably an aromatic or heterocyclic group, although aliphatic groupsor aroalkyl groups are included. Thus, a head group may be a fluorescentmoiety, which also serves as a “reporter.”

An “inhibitor” moiety or group is a chemical group derivatizing apolyamine that (1) causes the derivative to bind to the PATr with higheraffinity than does a native polyamine and/or (2) by other means blocksthe uptake of a polyamine (or a probe of this invention) into a cell ora subcellular PATr preparation. The inventors disclose herein compoundsthat efficiently inhibit PAT in MDA-MB-231 human breast carcinoma celland other cells. A number of different types of such inhibitors havebeen synthesized; various of the synthetic schemes are disclosed herein.

A “reporter moiety” is a chemical moiety forming part of a probe whichrenders the probe detectable (either directly or, for example, throughenzymatic enhancement) and hence permits the determination of theactivity of the PATr to which the probe binds. A reporter is detectableeither because it itself emits a detectable signal, or by virtue of itsaffinity for a reporter-specific partner which is detectable or becomesso by binding to, or otherwise reacting with, the reporter. In apreferred embodiment the polyamine analogue is immobilized to a solidsupport which enables removal of the analogue and anyinteracting/binding molecules from a complex mixture.

Overview of Structure-Activity Relationships (SARs)

The PAT inhibitors were developed by modification of the naturalsubstrate of the transporter, spermidine. The present inventorsdiscovered that introduction of a 3-amidopropyl group to thediaminobutyl part of spermidine produced a significantly bettertransport inhibitor as shown in FIG. 1. The optimal amido or sulfonamidesubstituent was found to be a medium sized aromatic group, leading tothe invention of N¹-dansylspermine (MDS) as both a transport inhibitorand a transport assay reporter molecule. MDS has increased bindingaffinity to cells compared to spermidine and N¹-acetylspermine.Significantly enhanced inhibition of cell growth and PAT resulted fromthe introduction of a 6-carbon atom linker between the aromatic “head”group of MDS and the polyamine core. This new molecule,N¹-[(N⁶-dansyl)-6-aminocaproyl]spermine (or DACS) 4, is one of the mostpotent PAT inhibitors known. In its interaction with biological systems,DACS shows many of the desired properties set forth above. The presentinventors have studied DACS and other related analogues extensively.

The SARs around DACS 4 as a lead compound have been explored extensivelyas shown in FIG. 2 (in particular, compounds 73-98). As discussed above,changes were made in each of several regions of DACS, and effects ontransporter binding were measured. The impact of changing the aromatic“head” group was explored by synthesizing a number a different activated4-nitrophenyl esters with different aromatic and non-aromaticN-sulfonamides at the distal amino end. Another series of “headless”analogues were synthesized to explore the importance of the hydrophobicaromatic grouping. In sum, the present inventors have designed andsynthesized a large number of compounds that efficiently inhibit PAT. Asdescribed herein, all mono, di and multi-substituted polyamines with thevarious substituents are intended for use as drugs.

A. N¹-Substituted Polyamine Analogues

A series of inhibitors was made by direct reaction of a polyamine with asulfonyl chloride, acyl, isocyanate, isothiocyanate, alkyl chloride oran N-hydroxysuccinamide-activated carboxy ester as described in FIG. 3and in Examples I-IV. Different head groups, linkages and polyamineswere combined. Many of the Figures show spermine as a nonlimitingexample of the polyamine core of the molecule.

The polyamine core can be varied as defined above. The synthesis ofN¹-(1-pyrenylsulfonyl)spermine 15 from spermine and 1-pyrenesulfonylchloride (FIG. 5) is described in detail in Example II.

The synthesis of N¹-((1-carbonyl)4-(1-pyrenyl)butane)spermine 7, fromspermine and pyrenebutyric acid (FIG. 6) illustrates the use of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (or EDAC) toform, in situ, the activated N-hydroxysuccinimide ester of a carboxylicacid. This one-step method produces the amide analogues of polyamines(see Example III). The synthesis ofN-(1-anthracenyl)-N′-(N¹-spermidyl)urea 9 from 1-aminoanthracene andspermine (FIG. 4) is described in more detail in Example IV. Thisillustrates the synthesis of ureas by activated urethanes asintermediates. Urea derivatives can also be synthesized usingsubstituted isocyanates. For example, 1-aminoanthracene is firstactivated with p-nitrophenyl chloroformate to form the urethane which isreacted with spermine to yield a substituted urea 9. The synthesis ofN-(N1-spermidyl)-2-(naphthyl)acetamide 103,N-(N¹-spermidyl)-2-(naphthoxy)acetamide 104 andO-(fluorenylmethyl)-N-(N1-spermidyl)urethane 105 are described inExample V-VII, respectively.

The best PAT inhibitors of this group have spermine as the polyaminecore and include a head group such as pyrenyl (see FIG. 5; Example II(15)), 5-(4-chlorobenzaamidomethyl)thiophenenyl (13) or dansyl (3) (FIG.7; Example I). These three compounds inhibit the PATr with K_(i)'s of91, 58 and 80 nM, respectively. A head group can also be attached tospermine via an amide bond as illustrated by compound 14, resulting in aK_(i) of 37 nM. Inhibitors of this type typically have K_(i) values ofapproximately 100 nM and R values in the MDA growth assay of >1.However, when spermine was substituted withN-(3-aminopropyl)-1,3-propanediamine,N,N′-bis-(3-aminopropyl)ethylenediamine,N,N′-bis(3-aminopropyl)piperazine,N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-bis(2-aminoethyl)-1,3-propanediamine, tris(3-aminopropyl)amine ortris(2-aminoethyl)amine, the K_(i) values in the polyamine transportassay were above 200 nM. Such less inhibitory compounds ate omitted fromFIG. 2 (which lists compounds 3-98). The synthesis of these types ofcompounds is exemplified in FIGS. 4-7 (Examples I-IV).

The Examples illustrate a key point regarding the synthetic methods. InExample I, the polyamine in CH₂Cl₂ solvent was treated dropwise to asolution of the acid chloride in the same solvent. This gave astatistical mixture of the unsubstituted, monosubstituted anddisubstituted polyamine derivatives, which is advantageous becausepurification by the methods described herein resulted in pure mono- anddi-substituted derivatives. Each analogue was then tested in thebiological assays (PAT inhibition and cell growth inhibition). It wassometimes an advantage to produce an individual mono-substitutedderivative using a mono-protected polyamine intermediate. Large-scale(>5 grams) production of the analogues was accomplished in this fashionbecause removal of side products was greatly facilitated.

The preferred mono-protected polyamine intermediates were the N¹-tBocderivatives produced according to Blagbrough et al., (Tetrahedron Lett.35:2057-2060, 1994), using di-tert-butyldicarbonate in tetrahydrofuran.Mono protected spermine was used to synthesizenaphthyl-2,6-bis(N,N′-spermidylsulfonamide) as described in Example VIII

B. Discovery of Lead Compound

Following structural explorations around the amide, sulfonamide or ureasubstituent, it was determined that introduction of a six carbon,straight chain aliphatic linker between the polyamine core and the headgroup led to a 10-fold increase in binding to the PATr (see FIG. 1).Given the high affinity this compound, DACS 4, to its biological target,it was selected as a lead compound for further modification. Two methodsfor the synthesis of DACS 4 are presented. The first method uses twocommercially available starting materials, appropriate for synthesizingsmall amounts of DACS 4. The synthesis DACS 4 from spermine and6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acidsuccinimidyl ester is shown in FIG. 8, (showing compounds 4, 99, 106)and described in more detail in Example IX The second, multistep method(FIG. 9; showing compounds 4, 99, 107-110), uses structurally flexiblesynthetic procedures for producing the modified analogues. The multistepproduction of DACS 4 in the second method (Examples X-XIII) illustratesthe procedure used to synthesize many of the linker analogues indescribed herein. This method is based in part on R. Goodnow et al.,(Tetrahedron Lett. 46:3267, 1990). The p-nitrophenyl ester of a N-tBocblocked amino acid is synthesized using DCC in EtOAc and then deblockedby the trifluoroacetic/CH₂Cl₂ method. The p-nitrophenylalkylaminoesteris then derivatized with an acyl chloride, sulfonyl chloride, or theequivalent, to introduce the head group. The N-substituted amino acidp-nitrophenyl ester reacts readily in methanol with excess polyamine toyield the desired product. The desired monosubstituted product ispurified from the excess polyamine and a minor di-substitutedside-product by low-pressure C18 reversed phase chromatography (RPLC)and CH₃OH/0.5N HCl elution. Alternatively, the product can be separatedon a weak cation exchanger such as BioRad®70, with a NH₄OH gradient. Amore detailed description is provided in Examples X-XIII. The twomethods shown in FIGS. 8 and 9 compare the two purification methods usedthroughout this work (Examples IX, XIII)

Using the second procedure, different “head” groups can be easilycoupled to the p-nitrophenyl activated ester (different “head groupsoutlined below). Following purification of this active ester, it can bereadily coupled to the various polyamine derivatives. This method alsogives great flexibility in the choice of linkers. Any compoundpossessing both an acid and an amino functionality can be incorporatedinto the molecule. See Examples IX-XIII.

Structural Modifications of DACS

The Polyamine Core

1. General Structural Issues

The structure below shows the general modifications that can be made tothe polyamine core of the compound.

where x, y and z vary independently and may be 0 to 12, and R₁, R₂, andR₃ may be H, alkyl or aryl group. Stereoisomers can be separated

A fruitful general approach to realize selectivity of binding to atarget (e.g., protein) of interest has been to synthesizeconformationally or stereochemically defined analogues of a bindingmolecule. By significantly reducing the number of possible rotomers orconformations a molecule can adopt, one can attain increased binding tothe desired site. Since the molecule no longer has to search the entire“conformational space,” its energy of interaction with the targetincreases many times.

Others have tried to solve the selectivity problem with polyamineanalogues by synthesizing conformationally restricted analogues. Ganemreplaced the butyl portion of spermine with 2-butene and 2-butynediamino derivatives (Ganem, B., J. Org. Chem. 1987, 52, 5044-5046).Rajeev, K. G. et al., J. Org. Chem. 1997, 62, 5169-5173, incorporated astereochemically defined, conformationally restrained pyrrolidine ringinto the spermine backbone (FIG. 10; 115, x=1) Brand, G. et al.,Tetrahedron Lett. 1994, 35, 8609-8612, synthesized cyclopolyamineanalogues of spermidine and spermine. See, for example FIG. 10 (113,x=3, 4, and 5). The present inventors extended this work by producingthe other analogues shown in FIG. 10. These analogues are synthesizedusing variations of known methods. The analogues where x=1 are producedby reacting spermine or N,N′-bis(3-aminopropyl)-1,3-propanediamine withformaldehyde as described by Ganem, B., Acc. Chem. Res., 1982, 15, 290).The primary amines are protected as N-tBoc derivatives for the analogues111 and 113. Acid deprotection then gives the desired products. Thederivative 112, where x=1, was also synthesized Ganem.

Analogues 111 and 113 (FIG. 10), where x=2 to 4, were produced byreductive alkylation. N¹, N¹⁴-Bis(tBoc)spermine was reacted with thedialdehyde, OHC(CH₂)_(x-2)CHO and NaBH₄ in EtOH. Compounds 112 and 114were made by the same procedure on a suitable N¹,N⁴-bisprotectedspermine derivative.

Stereochemically defined, internally cyclic structures (FIG. 10, 115)are synthesized using an intermediate aldehyde produced from alcohol 130shown in FIG. 4. This protected alcohol 130 can be oxidized to thealdehyde using Swern conditions. Aldehyde extension by the Wittigreaction with formylmethylene triphenylphosphorane, followed byreduction (overreduced alcohol can be reoxidized to the aldehyde usingpyridinium chlorochromate) and reductive amination/cyclization completedthe sequence to make the analogues where x=2. By Wittig reaction with3-bromopropyl triphenylphosphonium bromide, deprotection andintramolecular alkylative cyclization, the analogue where x=3 can beproduced. Either stereoisomer can be produced by starting with L- orD-ornithine. Polyamines containing a guanidinium group are synthesizedaccording to Iwanowicz, E. J. et al., Synthetic Comm. 23 1443-1445,1993.

2. Natural Polyamines

The natural polyamines, including putrescine, spermidine and spermine,are incorporated into the compositions of this invention by couplingthem to the various “head” and “linker” groups. Other naturallyoccurring polyamines that can be employed similarly include:N¹-acetylspermine, N¹-acetylspermidine, N⁸-acetylspermidine,N¹-guanidinospermine, cadaverine, aminopropylcadaverine, homospermidine,caldine (norspermidine), 7-hydroxyspermidine, thermine (norspermine),thermospermine, canavalmine, aminopropylhomospermidine, N,N′-bis(3-aminopropyl)cadaverine, aminopentylnorspermidine,N⁴-aminopropylnorspermidine, N⁴-aminopropylspermidine, caldopentamine,homocaldopentamine, N⁴-bis(aminopropyl)norspermidine, thermopentamine,N⁴-bis(aminopropyl)spermidine, caldohexamine, homothermohexamine andhomocaldohexamine.

3. N¹-Alkylated Polyamines

The metabolic stability in vivo of monosubstituted polyamine analoguesis increased by modifying these compounds to resist enzymaticdegradation. For example, substitution of the terminal primary aminegroup with an alkyl group would achieve this by preventing oxidativemetabolism. This invention also includes compounds with alkylatedsecondary amino groups. N-alkylation of the amide nitrogens slows downproteolytic degradation.

The foregoing changes can be achieved by a number of synthetic routes.Substitution of carbon atoms α to secondary nitrogens and acylation ofnitrogens can also slow degradation by polyamine oxidase. Such chemicalmodifications may minimize potential pharmacological side effects ofthese compounds.

To reduce potential metabolic degradation of derivatized PAT transportinhibitors, the terminal free primary amino group can be blocked byN-alkylation (Bergeron, R. J. et al., J. Med. Chem. 37:3464-347, 1994)as illustrated in FIG. 11 (compounds 2, 47, 77, 116-117). Lithiumaluminum hydride (LAH) reduction of N¹-acetylspermine 2 yields thedesired N¹-ethylspermine 116. Reaction of N¹-ethylspermine 116 orN¹-acetylspermine 2 with a N-substituted p-nitrophenylester of an aminoacid in methanol gives the desired compound modified with either anethyl or an acetyl group at the primary N¹.

Alternatively, methyl groups can be introduced α to the terminal aminogroups (121) of spermine (Lakanen, J. R. et al., J. Med. Chem.35:724-734, 1992). The 1,12-dimethylspermine analogue 121 was veryresistant to normal metabolic degradation. This compound is easilycoupled to a linker and head group as shown in FIG. 12 (compounds 66,18, 121). Ganem, B., J. Org. Chem. 1986, 51, 4856-4861, synthesized bisa-gem-dimethylpolyamine analogues. The present inventors have extendedupon these two reports and synthesized the bis-cyclopropylamineanalogues by the route described below. See FIG. 13. Reaction of theperbenzylated diamide with EtMgBr and Ti(O^(i)Pr)₄ according toChaplinski, V., Angew. Chem. Int. Ed. Engl. 1996, 35, 413-414 or Lee, J.J. Org. Chem. 1997, 62, 1584-1585 produced the fully protectedbis-cyclopropylamino analogue of spermine. Catalytic hydrogenationyields a fully deprotected polyamine. Other internally,cyclopropyl-substituted polyamine analogues can be produced in ananalogous manner to that shown in FIG. 13. Other analogues produced areshown at the bottom of FIG. 13. These cyclopropyl polyamine analoguesare activated by cellular enzymes to become alkylating agents.

Polyamine analogues of 4 with acetyl (4), N-ethyl (35) and α-dimethyl(66) substitution have been synthesized and shown to have K_(i)'s (forthe MDA-MB-231 cell PATr) of 2100, 41, 18 nM, respectively.

Detectably labeled polyamine derivatives can be synthesized usingradiolabeled ¹⁴C-spermine or other radiolabeled polyamine as startingmaterial.

4. Internally Substituted Polyamine Analogues

Various polyamine analogues alkylated at internal carbons can also besynthesized. 5-carboxyspermine, tetra tBoc-5-carboxyspermine and itsacid chloride are synthesized according Huber, H. et al., J. Biol. Chem.271:27556-27563, 1994. The resulting acid chloride can then be reactedwith various nucleophilic reagents to produce carboxy-substitutedpolyamine analogues following removal of the tboc group. These analoguescan then be coupled to the reagents that donate the linker and/or headgroup. Alternatively, the carboxy intermediate can be reduced to anintermediate that is used to synthesize numerous analogues. Suchanalogues are of interest in the present invention as alkylating agents(e.g., internal aziridine spermine derivatives) or as enzyme-activatedirreversible inhibitors of enzymes involved in polyamine biosynthesis,utilization and degradation (e.g., spermine synthase, deoxyhypusinesynthase, polyamine oxidase) as shown in FIG. 14 (compounds 130-134).Any enzyme that acts on the substituted carbon atom will generate ahighly reactive intermediate that can alkylate the enzyme's active siteresidues.

5. Commercially Available Polyamine Analogues

Many polyamine derivatives are available commercially, and these caneasily be derivatized further to make the polyamine analogues of thepresent invention.

Head Groups

1. General Description

The general construction of the lead compounds shown below indicates theconnections between the head group, linker and polyamine:

where coupler₁ is —C(═O)NH—, —S(═O)₂NH—, —NHC(═O)—, —HNS(═O)₂—,—HNC(═O)NH—, —HNC(═S)NH—, O—C(═O)NH—, —O—, —S—, —CH₂— or —NH—; and

coupler₂ is —C(═O)NH—, —S(═O)₂NH—, —HNC(═O)NH—, —HNC(═S)NH— or —NH—

A number of coupling chemistries can be used to combine the “head” groupand the linker moiety. Types of “head” groups are disclosed below as areadditional groups that can be substituted onto these head groups.

The coupling between the polyamine and linker will be described belowbefore description of the linkers. What follows is the definition of thehead groups.

The structural diversity of preferred head groups is very large, andmost organic groups that can be covalently attached to an amine arepotential candidates. The following table provides guidance regardingthe intended head groups but is by no means is intended to be limiting.Mono and multi-substitutions on the ring structures of these head groupsare also intended.

LIST OF HEAD GROUP SUBSTITUENTS halogen cyclohexyl ethoxyl propyl estermethyl cycloheptyl propoxyl isopropyl ester ethyl cyclooctyl thio cyanopropyl cyclononyl methylthio isocyanato isopropyl cyclodecyl ethylthiotrifluoromethyl butyl hexyl propylthio trichloromethyl isobutyl 2-hexylbutylthio tribromomethyl tert-butyl 3-hexyl isopropylthio azido pentylallyl nitro Acetoxy 2-pentyl vinyl amino Carboxamide 3-pentyl acetylenicacetamide N-methylcarboxamide neopentyl propargylic formamide N,N-dimethylcarboxamide cyclopentyl homopropargylic carboxylicN-ethylcarboxamide cyclopropyl hydroxyl methyl esterN,N-diethylcarboxamide cyclobutyl methoxyl ethyl ester

2. Aromatic Groups

Aromatic groups include phenyl naphthyl, 1-, 2-, or 3-biphenyl, indenyl,acenaphthylenyl, anthracenyl, phenanthrenyl, phenalenyl, triphenylenylpyrenyl, diphenylmethylenyl, etc.

3. Heterocyclic Groups

Heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl,morpholinyl, biphenyl, furanyl, pyrrolyl, 1,2-diazolyl, imidazolyl,1H,1,2,3-triazolyl, 1H-1,2,3,4-tetrazolyl, thiazolyl, oxazolyl,1,3,4-thiadiazolyl, pyridinyl, pyrimidyl, 1,2-diazinyl, 1,4-diazinyl,1,3,5-trizinyl, dibenzofuranyl, acridinyl, 2,1,3-benzothiadiazole,isoquinolinyl, quinolinyl, benzufuranyl, isobenzofuranyl,1,3-benzodiazinyl, phenazinyl, phenoxazinyl, phenothiazinyl, pyran,chromenyl, xanthenyl, indolizinyl, isoindolyl, indolyl, purinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,ptericinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,perimidinyl, phenanthrolinyl, isothiazoly, furazanyl, indolinyl,isoindolinyl, quinuclidinyl, and biotinyl.

4. Aliphatic Groups

This class includes straight-chain, branched and cyclic hydrocarbonsattached to the linker. The group includes C₂₋₁₀ alkanes; C₃₋₁₀ alkenescontaining 1 to 3 unsaturations; C₃₋₁₀ alkynes containing 1 to 3unsaturations; branched C₃₋₁₀ alkanes, alkenes and alkynes; polycyclicaliphatic hydrocarbons and steroid-like ring systems that include C₃₋₈cycloalkyl, adamantyl, camphoryl, cholesteryl, etc.

5. Miscellaneous-

a. DNA intercalators:

Coupling an intercalator to the polyamine will yield an agent with muchhigher affinity for nucleic acid targets. Examples of intercalatingagents amenable to this use are acridine, 9-aminoacridine, proflavine,actinomycin D, daunorubicin, doxorubicin, nogalamycin, menogaril,ellipticine, BD-40, amsacrine, acodazole, 2-pheylquinoline carboxamide,crisnatol, nitracrine, pyrazoloacridine, mitonoafide, ametantrone,mitoxantrone, oxanthrazole, bisantrene, echinomycin. For a review of DNAintercalating agents see Baguley, B. C., Anti-Cancer Drug Design 1991,6, 1-35.

b. Biochemical conjugates

Drug selectivity is achieved by targeting specific cells orenzymes/receptors on cells. The following biochemicals are candidatesfor coupling to polyamines for producing a selective pharmaceuticalagent: steroids, prostaglandins, phospholipids; enzyme cofactorsincluding nucleotide containing molecules such as NADH, AcetylCoA,AdoMet, flavin, tryptophantryptophyl quinone (TTQ), etc.

An additional series of head groups comprises polyamines conjugated topolyethylene glycol (PEG) or O-methylated PEG (abbreviated MeOPEG)polymers of various sizes.

6. Multiple Ring Head Groups

Head groups can vary from simple alkyl substitutions to multi-ring andmulti-single-ring substitutions. Some of the structural variations areschematically represented in FIG. 15.

Spacers X, Y and Z (for example FIG. 15, compounds 135-139) are definedas bonds or straight chain groups that attach different ring structuresin a multiple ring head group. In some cases the spacers function asdirect C—C or C—N attachments. Conventional spacers known in the art aresimilar to the linkers described herein. Known chemistries are used forcovalent attachment of a ring structure in a head group with a spacer,for example, the formation of amide, sulfonamide, ether, thioether,ester, —C—C— and —C—N— and —N—N—bonds. R₁, R₂ and R₃ are typicallyalicyclic, aromatic, or heterocyclic rings when substituted inmulti-ring head groups. These ring structures individually can also besubstituted. Some of the multi-ring head group types described above areavailable from commercial sources, and examples are shown as structures140 to 147 in FIG. 16. Alternatively, these or similar compounds arereadily synthesized.

Linker Group

1. General Description

The linker portion of the compound can be represented by a generalstructure with an amino group at one end and an acid group on the other.One group of linkers contains diamino groups that are bonded via a urealinkage to the polyamine and via an amide, urea or sulfonamide linkageto the head group. The head group can also be bonded through othercouplings such as ether, thioether and C—C bonds. The schematicstructure shown above (in the section labeled “Head Groups, 1. GeneralDescription) shows the function of the linker moiety connecting the headgroup to the polyamine and possessing a desired length and combinationof steric, conformational and hydrophobic properties. Also shown are thepossible combination of coupling methods. Each coupling method can beused in combination with any of the three methods in FIG. 3 at the otherposition to result in a wide array of desired properties.

The linker group can have a range of properties that are reflected bythe number of variations discussed below. Changes in the linkerstructure will be affect the properties of the whole polyamine analoguesuch as hydrophobicity, hydrophilicity, distance between head andpolyamine portions, steric arrangement of head and polyamine portions,conformational properties, solubility and electronic properties.

2. Aliphatic Straight Chain Linkers

A series of linkers was been synthesized to test the effect of differentdistances between head group and polyamine. This series is most simplyrepresented by the straight-chain aliphatic linkers having variouscarbon chain lengths shown below as compound 148).

The present inventors discovered that linker length had dramatic effectson the PAT inhibitory activity and the cell growth inhibitory activity.A low K_(i) is optimal for C₆ linkers in the presence of an aromatichead group. However, in the absence of a head group, differences ingrowth or transport inhibitory activities have not been dramatic. Thus,“headless” compounds have K_(i)'s in the order of about 25 nM but havemore attenuated inhibitory effects cell growth (breast cancer cell line)most likely due to their ability to actually be transported. Theprostate cancer cell line is more powerfully inhibited by these“headless” inhibitors as shown in FIG. 18 and Example XI. TheC3-headless compound had dramatic effects on cell growth.

The synthetic route to this series of compounds, starting with variouspolyamines and head groups, is represented by the DACS 4 syntheticscheme depicted in FIG. 9 and discussed in more detail in Example IX toXIII). The amino group is protected by the N-tBoc group, and thecarboxylic acid is then activated by forming the p-nitrophenyl ester.After acid deprotection of the N-tBoc group, the amino group can bereacted with an acid or sulfonamide chloride of the desired head group.After purification, direct reaction with the polyamine of choice inmethanol gives the desired product. This can be purified by either (1)reverse-phase silica gel chromatography using 2:9 MeOH/0.5 N HCl or (2)cation-exchange chromatography over BioRex 70 resin (NH₄ form) using alinear gradient of from 0 to 2N NH₄OH.

3. Unsaturated Straight-chain Aliphatic Linkers

Varying degrees of unsaturation (alkene and alkyne) together with thegeometric isomers of the alkene derivatives can be introduced into thelinker moiety as depicted below (149 and 150). These variations allowintroduction of conformational restraint into the final product.

where n=0 to 7 and m=1 to 4

4. Carbon-substituted and Cyclic Aliphatic Linkers

Branched chain and cyclic saturated aliphatic linker groups imposeconformational restraint on the desired polyamine analogue. Compounds151 and 152 below illustrates this class of structure.

where n=1-10; R and R′ vary independently and can be H or CH₃(CH₂)_(m),and where m=1 to 10.

5. Chiral Carbon-substituted Amino Acid Linkers

Great structural diversity can be incorporated quickly into thepolyamine analogues by using any of the large number of chiral aminoacids that are available commercially. Many of the chiral amino acidintermediates to be used in the synthetic scheme shown in FIG. 9 arealso available commercially, including some N-tBoc protected amino acidsand some N-tBoc protected amino acid p-nitrophenyl esters. FIG. 19 (153)illustrates a variety of derivatives that have been produced by thismethod.

An additional thousand α-amino acid analogues known in the art can beused to form polyamine adducts. These are very easily incorporated intothe present invention through the synthetic sequences described in FIGS.8 and 9. Several key examples are; t-butylglycine, ornithine,α-aminoisobutyric acid, 2-aminobutyric acid, α-aminosuberic acid,4-chlorophenylalanine, citrulline, β-cyclohexylalanine,3,4-dehydroproline, 3,5-diiodotyrosine, homocitrulline, homoserine,hydroxyproline, β-hydroxvaline, 4-nitrophenylalanine, norleucine,norvaline, phenylglycine, pyroglutamine, β-(2-thienyl)alanine, etc.Several important β-amino acids are easily incorporated into the presentinvention through the chemistry discussed above. A key example isβ-alanine, etc.

Both stereoisomers of the natural L-amino acids (L═S) or D-amino acids(D═R) can be used in this invention. Because each isomer can be usedindividually, the structural diversity of the analogues is markedlyenhanced.

6. “Headless” Linkers

The desired biological properties do not always depend upon the presenceof a head group. Hence, a large series of so-called “headless”derivatives, containing a polyamine and linker without a head group weresynthesized and tested. These derivatives are made by reacting theactive ester (p-nitrophenyl or N-hydroxylsuccinimide) of the N-tBocamino acid with the polyamine of interest. The resulting N-tBocprotected derivatives are then purified by cation-exchangechromatography over BioRex 70 (NH₄ form) resin using a linear gradientfrom 0 to 2N NH₄OH. The tBoc group can then be cleaved by acidtreatment. Both the tBoc and acid deprotected derivatives can be testedfor biological activity. The full series of amino acids discussed above,together with other derivatives have been synthesized. A more detaileddiscussion of the synthesis of N¹-[6-aminocaproylspermine] appears inExample XIV.

Reactive, Irreversible Polyamine Transport Inhibitors

A. Alkylating Reagents-

1. Aziridines

Polyamines substituted with fluorophores and other bulky end group werefound to have the intrinsic property of high avidity binding to thePATr. This suggested that, in addition to utility as a diagnostic orresearch tool, they are useful as therapeutic agents for treatingdiseases or conditions wherein it is desirable to inhibit PAT. Theirintrinsic affinity for other polyamine targets such as DNA broadens evenfurther the scope of their therapeutic utility.

In a preferred embodiment the polyamine core is substituted with theaziridinyl group. The embodiment shown in FIG. 20 has a secondsubstituent (a fluorophore such as dansyl or another bulky group).Aziridinyl-substituted polyamines react with nucleophilic groups intarget binding complexes (receptors, transporters, enzymes and nucleicacids). In addition they can be exploited to bind other reactivemoieties to polyamines. These mono- and di-substituted polyamineanalogues are useful as drugs because of their inhibition of (a) thePATr, (b) polyamine synthesis and (c) reactions that use nucleic acidsas substrates.

In one embodiment, a reactive group other than aziridine is introducedinto a polyamine already substituted with a head group and a linker.This reactive group allows the labeled polyamine to bind covalently toan appropriate nucleophilic site on a polyamine-binding target moleculesuch as the PATr. Compounds of this type are used to covalently labelreceptors, enzymes or nucleic acids; thus, the modified polyamine servesas an affinity label that is useful in diagnostic assays and as a toolto isolate a polyamine binding target. Again, such compounds used asdrugs will treat diseases or conditions which are ameliorated byblocking PAT or DNA-polyamine interactions. By virtue of the relativeirreversibility of their binding, such compounds can be used at lowerdoses or at decreased frequency compared to compounds known in the art.

Disubstituted polyamines are synthesized by using the appropriate amineprotecting groups on the polyamines. Reagents for the stepwisefuctionalization of spermine are known (Bergeron, R. J. et al., J. Org.Chem. 53: 3108-3111 (1988); Byk, G. et al., Tetrahedron Lett. 38:3219-3222 (1997)). Bergeron et al. (supra) described the use of fourindependent amine-protecting groups: benzyl, t-butoxycarbonyl,trifluoroacetyl, and 2,2,2-trichloro-t-butoxycarbonyl. Conditions thatallow the selective removal of each protecting group were alsodescribed. These reaction conditions allow independent and selectivederivatization of each nitrogen of spermine. Thus this inventionincludes derivatization of monofunctionalized spermine with alinker/head group on any one of the four nitrogens and the synthesis ofpolyamine analogues with more than one functionalized nitrogen.

Methods to introduce an aziridine group into spermine (Li et al, J. Med.Chem., 39:339-341 (1996) and into derivatives of spermidine (Yuan et al,Proc. Am. Assoc. Cancer Res., 34: 380 (1993) are available. A syntheticscheme for N¹-(aziridinyl)-N¹²-[(N⁶-dansyl)-6-aminocaproyl]spermine isshown in FIG. 20 (154-157).

Whereas FIG. 20 shows the synthesis of the spermine derivative, anyother polyamine derivative can be produced using an appropriatelyprotected polyamine precursor, coupling to the linker/head group moietyand reductive amination with 3-aziridinepropanal. Removal of theprotecting group(s) then gives the desired, reactive polyaminederivative. An additional example of this approach, illustrating thechemical flexibility it permits, is shown in the FIG. 21 (158-160).

3. Other Reactive Groups

Other useful moieties that can be added instead of the aziridine groupand that react with nucleophiles to form covalent bonds include chloro-,bromo- and iodoacetamides, sulfonylfluorides, esters, nitrogen mustards,etc.

The chemically reactive 2-haloacetamide group can easily be introducedinto any of the polyamine analogues by reaction with the appropriate2-haloacetic acid halide. Other chemically reactive groups are describedbelow.

B. Photochemically Activated Reagents

The use of photochemically activated functionalities on biologicallyactive molecules is a well known (Fleming, S. A., Tetrahedron51:12479-12520, 1995). In the polyamine field, Felschow et al. attachedan azidobenzoic acid moiety to spermine and examined the interaction ofthe resulting adduct with cell surface proteins (Felschow, D M et al.Biochem. J. 328, 889-895, 1997; Felschow, D M et al., J. Biol. Chem.270:28705-28711, 1995). Since their photoprobe had an apparent K_(i) of1 μM versus spermidine for the PATr, the photolabelled proteinsdescribed were a mixture of polyamine binding proteins. One of the mostpotent PAT inhibitors of the present invention, DACS, has a Ki of <10nM, which indicates an affinity 100 times higher than the compoundreported by Felschow et al. Therefore introduction of a photoactivatablegroup to this molecule holds great promise in the isolation of the PATrprotein(s).

1. Azide

Substitution of the dimethylamino group in dansyl chloride by azideproduces a photochemically reactive chemical group. The preparation of1-azido-5-naphthalene sulfonyl chloride has been described (Muramoto,K., Agric. Biol. Chem., 1984, 48 (11), 2695-2699), and it is alsoavailable commercially from Molecular Probes Inc. (Eugene, Oreg.).Introduction of this compound into the synthetic scheme for DACS isstraightforward and merely requires substitution for dansyl chloride.

This azido derivative, would enable isolation and characterization ofthe PATr protein(s), and would also find use as an irreversible,photoactivatable drug molecule.

2. Diaziridines

Substitution of a diaziridine group on the head group would accomplishmany of the same goals as noted above.

3. Diazo Groups

Polyamine analogues with photoactivatible head groups are made usingp-nitrophenyl 3-diazopyruvate, a reagent for introduction of aphotoactivatable 3-diazopyruvate group to an aliphatic amine. This agentis also available from Molecular Probes, Inc. The desired derivative ismade by reacting this reagent with the free amino, p-nitrophenylactivated linker precursor, purifying the linker/head groupintermediate, and reacting it with the polyamine.

Reporter Molecules (Probes ) for PAT and Other Polyamine-bindingProteins

Various moieties can be attached to polyamines to produce novelinhibitors of PAT with utility as probes in a PAT assay and for drugscreening. Such chemical modifications do not destroy effective bindingand, in fact, enhance the affinity of the derivatized polyamine for thePATr. Such compounds are thus useful for measuring polyamine uptake and,more importantly, in high throughput screening assays to discovertherapeutically useful inhibitors of this uptake.

In a preferred embodiment the polyamine analogue is immobilized to asolid support which enables removal of the analogue and anyinteracting/binding molecules from a complex mixture.

Because a number of polyamine binding protein are targets fortherapeutic intervention, an improved assay for polyamine binding oruptake is desirable. Small changes in polyamine structure can havedrastic effects on activity. Reporter molecules are polyamine analoguesthat are conveniently detectable while maintaining their bindingactivity to the PATr. For optimal binding to the PATr, the reporters aresufficiently specific so that binding other polyamine binding moleculesdoes not interfere in the assay. N¹-substituted polyamines arecompetitive inhibitors for the PATr and for several other polyaminebinding proteins such as the external sensing Ca⁺² receptor and the NMDAreceptor. There is no report in the art of a molecule that binds to thetransporter (or any other polyamine binding membrane receptors) with thehigh affinity of the present probes. The inventors have thereforefocused on producing suitable reporter-bound polyamines that maintain areasonable range of detectability and that bind competitively withspermine. FIG. 1 illustrates a synthetic scheme for various N¹-linkedpolyamine analogues.

Reporter Head Groups

The Head/Reporter group structural diversity is very large and includesmost organic groups that can be covalently bonded to an amine. Thegroups described below are but several examples of the head groups ofthe present invention and are not intended to be limiting.

A. Fluorescent/Chemiluminescent Head Groups

The dansyl fluorescent group can be substituted by any of a large numberof fluorophores, for example, as disclosed in Haugland, Handbook ofFluorescent Probes and Research Chemicals, Sixth Edition, MolecularProbes, Eugene, Oreg., 1996. A number of dansyl-polyamine-like moleculesare known and commercially available, including: didansylcadaverine,monodansylcadaverine, didansylputrescine, MDS and tridansylspemidine(Sigrna Chemical Company, St. Louis, Mo.). The syntheses ofmonodansylputrescine (Raines, D. E. et al., J. Membrane Biol.82:241-247, 1984) and monodansylcadaverine (Nilsson, J. L. G. et al.,Acta Pharm. Suedica 8:497-504, 1971) have been described.

In general, a fluorescent reagent is selected based on its ability toreact readily with an amino function of a polyamine such as spermine.Examples of such fluorescent probes include the Bodipy™(4,4-difluoro-4-bora-3a,4a-diaz-s-indacene) fluorophores which span thevisible spectrum (U.S. Pat. No. 4,774,339; U.S. Pat. No. 5,187,288; U.S.Pat. No. 5,248,782; U.S. Pat. No. 5,274,113; U.S. Pat. No. 5,433,896;U.S. Pat. No. 5,451,663). The preferred member of this group is4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid.Preferred fluorophores for derivatizing polyamines according to thisinvention are those which are excited by ultraviolet light. Suitablefluorescent materials include Cascade Blue™, coumarin derivatives,naphthalenes (of which dansyl chloride as exemplified herein is amember), pyrenes and pyridyloxazole derivatives, Texas red™, Bodipy™,erythrosin, eosin, 7-nitrobenz-2-oxa-1,3-diazole (NBD), pyrenes,anthracenes, acridines, fluorescent phycobiliproteins and theirconjugates and fluoresceinated microbeads. Use of certain fluors such asthe phycobiliproteins and fluoresceinated microbeads will permitamplification of the fluorescent signal where cells have few PATr sites.

Fluorescein, fluorescein derivatives and fluorescein-like molecules suchas Oregon Green™ and its derivatives, Rhodamine Green™ and RhodolGreen™, are coupled to amine groups using the isocyanate, succinimidylester or dichlorotriazinyl-reactive groups. The long wavelengthrhodamines, which are basically Rhodamine Green derivatives withsubstituents on the nitrogens, are among the most photostablefluorescent labeling reagents known. Their spectra are not affected bychanges in pH between 4 and 10, an important advantage over thefluoresceins for many biological applications. This group includes thetetramethylrhodamines, X-rhodamines and Texas Red derivatives.

In yet another approach, an amino group or groups in the polyamine arereacted with reagents that yield fluorescent products, for example,fluorescamine, dialdehydes such as o-phthaldialdehyde,naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.7-nitroben-2-oxa-1,3-diazole (NBD) derivatives, both chloride andfluoride, are useful to modify amines to yield fluorescent products.

Those skilled in the art will recognize that known fluorescent reagentsmodify groups other than amines. See for example, Haugland, R. P.,supra; Chapter 2, where modification of thiols is described at pages47-62. Modification of alcohols, aldehydes, ketones, carboxylic acidsand amides is described in Haugland, supra (at pages 63-81). Hence,fluorescent substrates for the PATr can readily be designed andsynthesized using these other reactive groups. The most preferredreporter moiety is a fluorophore, either one which phosphorescesspontaneously or one which fluoresces in response to irradiation withlight of a particular wavelength.

Chemiluminescent reporters are also acceptable. Examples of particularlyuseful chemiluminescent labeling compounds are luminol, isoluminol,theromatic acridinium ester, imidazole, acridinium salt and oxalateester.

B. Radioactive Head Groups

In order to use a probe for detection, various radioactive groups can beadded for localization of the probe. Radioactive groups can be any ofthe various sulfonyl chlorides, acyl halides, or other activated groupsor non-activated groups that contain a radioactive element which can beincorporated into polyamine analogues. The polyamine itself can containa radioactive element which may aid in detection.

Radioactive reporter moieties are detected by measurement of the emittedradiation. Examples of suitable radioactive reporter moieties are thoselabeled with gamma -emitters such as ¹²⁵I, ¹²³I and ^(99m)Tc. Some α andβ emitters may suffice for detection.

C. Immobilizable Head Groups

With the large supply of various antibodies that are commerciallyavailable and the variety of conjugating systems like avidin-biotin, itis possible to place various immobilizable head groups into thepolyamine analogue. These include small molecules for which specificantibodies are commercially available, such as anti-dansyl,anti-fluorescein, anti-BODIPY, anti-Lucifer Yellow, anti-Cascade Blueand anti-DNP. Proteins or peptides can also be derivatized to polyaminesfor detection with available antibodies.

D. Solid Support

In various embodiments of the enzymatic assays, the immobilization maybe to any “solid support” capable of binding a capture protein (e.g.,streptavidin or antibodies). Well-known supports, or carriers, includeglass, polystyrene, polypropylene, polyethylene, polyvinylidenedifluoride, dextran, nylon, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have any structuralconfiguration so long as the immobilized molecule is capable of bindingto its ligand. Thus, the support configuration may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube ormicrowell, the external surface of a rod, or a chip. Alternatively, thesurface may be flat such as a sheet, test strip, microwell bottom, etc.Also useful for certain embodiments are magnetizable groups such asgadolinium complexes or electron-opaque groups. Those skilled in the artwill know many other suitable carriers for binding antibody or antigen,or will be able to ascertain such by use of routine experimentation.

Assay of Polyamine Transport

The preferred embodiment of the PAT assay for identifying PAT inhibitorsis a high throughput assay using a fluorescent polyamine-like probe.Transport of the probe through the cell membrane via a PATr is tested inthe presence of candidate transport inhibitors. This and all subsequentsteps are preferably performed by a robotic system for increasedefficiency.

Cells are plated in 96 well sterile microplates at an appropriatedensity for adherence and mid-log growth within 15-96 hours (the “testplate”). For an initial rate measurement, a pre-plate (96 well plateformat) is prepared using, for example, a combinatorial library ofpotentially inhibitory compounds (such as those prepared by PanLabsInc., Bothell, Wash.). See also description of plyamine combinatoriallibraries, below.

In addition to 1 nmole of the test inhibitor in each well, 1 μmole ofaminoguanidine and 1-100 nmoles of the fluorescent polyamine probe areadded. The final volume can be from about 25 μl to about 200 μl.Aminoguanidine is included to prevent the oxidative breakdown of thepolyamine compounds in the culture medium.

The assay is initiated by transferring the contents of the pre-plate tothe test plate in a timed fashion. The cells are incubated with the testinhibitor and the fluorescent polyamine probe for 1-60 minutes. Theincubation is terminated by removing the medium and washing the wellsthree times with ice cold medium containing 1 mM aminoguanidine and 1 μMspermidine. The cells are then lysed with detergent (e.g., 100 μl of0.1% sodium dodecylsulfate) and transferred to a fluorescence platereading system, preferably a top reading fluorescence spectrophotometer.

A well is scored as positive if the test inhibitor causes thefluorescence to drop below 50% of the negative (no inhibitor) control.The inhibitor compounds which score positive are then tested todetermine their binding constants by repeating the above assay whilevarying the concentrations of both the fluorescent probe and the testinhibitor. A standard kinetic analysis is performed to quantify the typeof inhibition (e.g., competitive vs. noncompetitive) and itseffectiveness.

Using a conventional radiometric assay (described below), the presentinventors were able to screen about 20 compounds per week for theability to inhibit PAT in a cell line. With the high throughputfluorescent assay disclosed herein, one can screen 250 compounds or moreper week. Although the radiometric assay might lead to identification ofthe same compounds, the time scale would be much longer.

As described, the preferred detection method is based on thefluorescence of the modified polyamine probe of the present invention.However, there are a number of other useful detection methods whichinclude chemiluminescence, colorimetry as well as conventionalradiometry. For chemiluminescence detection, a chemiluminescent group issubstituted for the fluorescent group. For example, fluorescentfluorescamine adducts can be converted to chemiluminescent products withbis-trichlorophenyl oxalate (Walters, D L et al., Biomed. Chromatogr.8:207-211, 1994). In yet another embodiment, calorimetric detection isused with chromophores having high extinction coefficients.

High throughput screening (HTS) has become an essential part of therapid drug discovery process. Using an HTS assay, many compounds can beassayed using candidate compounds from existing libraries or librariessynthesized by combinatorial approaches such as that described herein.Compounds numbering in the thousands to hundred thousands are nowroutinely screened in the pharmaceutical industry, using microtiterplate formats with either 96 or 384 wells and robotic devices whichtransfer reagents, wash, shake and then measure activity signals whichare directly imported in computer compatible form. The robotic devicesand optimization programs to aid rapid drug development are well-knownart. The three requirements of HTS are speed, accuracy and economy.

A. Radiometric Assays

In a conventional radiometric assay, the cells are incubated undergrowth conditions with [5,8-¹⁴C]spermine in serum-free medium forvarying intervals. Cells are plated at a known density in 24 well platesin standard tissue culture medium and allowed to adhere and grow for15-96 hours. Due to the low signal of the radiolabel in this assay, 96or 386 well microplates cannot be used in this assay, creating a majorbottleneck for throughput. Cell numbers are determined, and the plate isplaced in a temperature controlled system at 37° C. Aminoguanidine isadded to the medium to a final concentration of 1 mM. The inhibitor tobe tested and the radioligand (preferably ³H-spermidine, ¹⁴C-spermidineor ¹⁴C-spermine) are prepared in separate plates. The assay is initiatedby the mixing of the inhibitor and radioligand. The cells are incubatedfor an interval of about 1-60 minutes depending on cell type. The assayis terminated by removing the medium and cooling the plates to 4° C. Thecells are then washed with cold medium three times, dissolved in 0.1%sodium dodecylsulfate, and the radioactivity in solution is determinedby scintillation counting.

B. Development of Polyamine Analogues for Use with PAT Assay

As indicated above, reporter moiety can be bound to a terminal amine orto an internal site of a polyamine. Most preferred are singlesubstituted fluorescent groups bonded to any of the nitrogens.Substitution of fluorescent groups on the different carbons of apolyamine is exemplified by N-(4-dansylaminobutyl)spermine-5-carboxamide. The number of reporter groups, e.g.,fluorophores, per polyamine analogue may vary. The maximum number offluorophores-that allows competitive interaction with the PATr ispreferred. In the case of monodansylspermine (MDS), the optimal numberof fluorophores is one. If the reporter moiety is dansyl, thesensitivity of the assay can be improved by use of an anti-dansylantibody (Molecular Probes, Eugene, Oreg.) during fluorescencemeasurement (in accordance with the manufacturer's instructions).

Preferred polyamine probes for the PAT assay have the followingcharacteristics: they bind to the PATr, compete with the naturalsubstrate (FIGS. 27 and 28) and are internalized into the cell aftersuch binding (Example XXV). A preferred probe comprises a fluorophore, achromophore or a luminescer such as a chemiluminescer or a bioluminescer(Clin. Chem. 25:512, 1979) coupled to a polyamine core.

The amount of probe taken up in an assay, or the intracellularlocalization of the probe are determined by measuring and/or localizingthe signal emitted by the reporter moiety after the probe has had theopportunity to bind to the PATr and be taken up. The probe may be aspecifically binding ligand or one of a set of proximal interactingpairs.

C. Enzymatic Polyamine Transport Screening Assay

1. Enzymatic Enhancement ELISA

Another preferred approach to a sensitive screening assay uses theenzymatic amplification of the signal emitted by the detectable label onthe polyamine probe. A more specific example is a cofactor-labeled probewhich can be detected by adding the enzyme for which the label is acofactor and a substrate for the enzyme. Such an enzyme is preferablyone which acts on a substrate to generate a product with a measurablephysical property such as color. Examples of such enzymes are listedbelow.

2. Polyamine Immobilization for Enhanced Detection

A preferred example of an enzymatically enhanced assay may be done byexploiting the biotin/streptavidin system (see FIG. 31). A biotinylatedpolyamine such as spermine is prepared, for example, as shown in FIGS.29 and 30. The PAT assay is performed as described above except that theprobe in the appropriately lysed cells is detected using one of theformats described below. For example, after allowing cells to transporta biotinylated polyamine (e.g., N¹-biotinylspermine), the cells arelysed and the lysate filtered through negatively charged microplatemembranes which retain the positively charged polyamine. After washesand appropriate blocking of unreacted sites with a protein such asalbumin, the membranes are incubated with a streptavidin-enzymeconjugate and washed. The membranes are treated with a chromogenicsubstrate of the enzyme and incubated for a set time after which thecolor signal is read in a spectrometer. Colorimetric, fluorescent andchemiluminescent substrates are compatible with this procedure.

In another embodiment, the lysate is reacted with a polylysine-treatedplate using glutaraldehyde to crosslink the polyamine analogue to theplate. The Schiff base is then reduced to produce an immobilized ligandor analogue such as DACS. The ligand can then be detected through theappropriate reporter system. In the case of DACS, the reporter isdetected by anti-dansyl IgG antibody coupled to an anti-rabbit IgGconjugated to an HRP (horse radish peroxidase) detection system (FIG.32; Example XXVI). In another embodiment, a fraction obtained from thelysate, for example using Sepharose S or Cibacrom Blue, is used.

The advantages of this procedure are (1) the immobilization of thebiotinylated spermine to the membrane allows the removal of interferingcompounds through washes, and (2) the amplification through enzyme- orantibody-coupled reporter systems increases sensitivity compared withsingle labeled substrates. The increased sensitivity requires fewercells per assay. The microtiter plate format allows for rapid HTS.

An additional embodiment of the enzymatically enhanced assay is the useof an antibody to the reporter moiety such as the hapten2,4-dinitrophenyl group (DNP), 2,4,6-trinitrophenyl (TNP), or,preferably, dansyl. Alternatively, the polyamine can be conjugated withdifferent hapten groups (other than biotin). In the case of biotinylatedspermine derivatized with dansyl, after transport into the cells andcell lysis, these molecules are immobilized to streptavidin-coatedmicroplate wells and washed. An antibody specific for dansyl is allowedto bind to any dansyl groups which have become immobilized. Theimmobilized antibody is then allowed to react with a second antibody (ananti-immunoglobulin specific for the anti-dansyl antibody) conjugated toan enzyme. After the second antibody has been allowed to bind to theimmobilized complexes, a chromogenic substrate for the enzyme is addedfor a set interval. The evaluation of the enzyme-generated signal (colorreaction) is a measure of the amount of polyamine bound to the PATr.

The foregoing enzymatic assays are in principle, similar to well-knownenzyme immunoassay or enzyme-linked immunosorbent assay (ELISA). See,for example, Butler, J. E. (ed.) Immunochemistry of Solid PhaseImmunoassay, CRC Press, Boca Raton, 1991; Van Regenmortel, M. H. V. (ed)Structure of Antigens, Vol. 1 ( CRC Press, Boca Raton 1992, pp.209-259). Butler, J. E. et al., J. Immunol. Meth. 150:77-90, 1992;Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,1980). Enzymes which can be conjugated to the streptavidin or antibodyinclude, but are not limited to, horseradish peroxidase, alkalinephosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase,staphylococcal nuclease, Δ-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease,urease, catalase, glucoamylase and acetylcholinesterase.

Immobilization of a Second Head Group for Enhanced Detection

In another embodiment, the biotin-spermine-hapten (preferably dansyl)molecules are captured using an immobilized anti-dansyl antibody. Inthis instance, two reporter molecules must be coupled to the polyamineanalogue as in FIG. 31. One reporter is used for immobilization such asto an antibody-containing bead (or other solid support) to enable thewashing away of excess probe. The other reporter is used for sensitivedetection. Streptavidin conjugated to an enzyme or streptavidin followedby enzyme-conjugated anti-streptavidin is preferred for signalamplification/detection.

The linker between biotin and the polyamine is important. It should bedesigned to maximize binding with steptavidin after the polyamine-biotinhas been immobilized to the solid support (Affinity Chromatography:Principles and Methods, Pharmacia, 1993). A number of different linkermolecules, generally commercially available, can be introduced betweenthe polyamine and biotin, e.g., biocytin. Many different linkers ofvarying lengths are known in the art. The same considerations areimportant for ligands/labels other than biotin.

Assays of Other Pharmacological Targets Using Polyamine Probes

The present invention may also be used for histochemical or cytochemicallocalization of polyamines after uptake. The polyamine analoguesdescribed herein localize within the cell. Probes of this type cantherefore be used in cytochemical analyses of cells or tissues toidentify cells or sites within cells with abnormally high or low levelsof polyamines. N¹-dansylspermine was shown to localize specifically tothe nucleoli and the nuclear membrane (Example XXVI). The structure ofthe nucleus is a known indicator for the staging of progressing cancer.The present fluorescent probes can be incorporated into traditionalcytological analysis with the use of a fluorescence microscope toenhance the accuracy of current diagnostic techniques.

In addition to the PAT activity assay, the reporter probe can be used toquantitate the binding of polyamines to known polyamine targets andbinding sites. These sites include the NMDA receptor, K⁺ inwardlyrectifying channel (IRK), protein kinase CK2, and phospholipase Cδ1.Polyamines also bind specifically to RNA and DNA, and polyamineinteractions play a role in hypusine synthesis. Due to the ease ofdeveloping modified polyamine analogues as described herein, specificanalogues can be developed for each polyamine binding target.

Soluble Proteins that Bind Polyamines

Many proteins bind polyamines. Assays for such proteins are included inthe scope of this invention and can identify drug candidates bycompetition assays using a bound fluorescent polyamine. In addition atightly or irreversibly binding polyamine analogue can be used toextract and isolate any polyamine-binding protein or otherpolyamine-binding target of interest. Some proteins undergoconformational changes when a substrate or a polyamine is bound. Thepresent screening assays are adapted so that the probe either binds ordoes not bind to a protein when the latter is in a given conformationalstate. One example is Protein Kinase A2, an isozyme that, upon bindinpolyamine, undergoes a conformational change that modulates the enzyme'ssubstrate binding site. An isozyme of methionine adenosyltransferase(MAT2) that comprises only the α subunit is much more sensitive topolyamine inhibition than the isozyme having the α and β subunits.

Testing Inhibitors of Polyamine Transport

Through screening compounds made by the various synthetic routesdescribed above, several compounds were found to effectively inhibitpolyamine transport. DACS 4 is one such compound, with a K_(i) 10 nM. Toreinforce its effectiveness as a PAT inhibitor, DACS was tested as aninhibitor of cell growth (FIGS. 22-24; Example XIX) in the presence ofpolyamines or an ODC inhibitor. DFMO. R values were calculated as theratio of the IC₅₀ in the absence of DFMO over the IC₅₀ in the presenceof DFMO (Example XX). Using both a kinetic measure and a biologicalassay, the present inventors observed high correlation between theinhibition of PAT and growth. The three compounds 6, 4 and 5 in FIG. 2(Example XX) had the best combination of K_(i)'s (5, 10 and 10 μM,respectively) and R values (220, 400 and 210, respectively) assummarized below:

Inhibitor Ki (μM) R 6 5 220 4 10 400 5 10 210

Several other compounds unrelated to polyamines were shown to inhibitPAT by a non-competitive mechanism. These compounds (FIG. 25) includeseveral anti-psychotic drugs (trifluoperazine and thorazine). Compounds161 and 162 had PAT inhibitory activity (see Example XXI). Compound 163,previously shown to be a PAT inhibitor, is also and antipsychotic drug.

Example XXI describes the inhibition of spermidine/spermineacetyl-transferase enzymatic activity by DACS (FIG. 26). Based on this,some of these compounds, if internalized, may serve a dual purpose.

The effect of various “headless” polyamine analogues were also evaluatedand are described in Example XXIII.

Pharmaceutical and Therapeutic Compositions

Preferred compounds for use in pharmaceutical compositions include allof those mono- and di-substituted polyamine compounds described above,most preferably DACS (4) and compounds 5, 171 and 6, primarily in theform of pharmaceutically acceptable salts of the compounds.Pharmaceutically acceptable acid addition salts of the compounds of theinvention which contain basic groups are formed where appropriate withstrong or moderately strong, non-toxic, organic or inorganic acids inthe presence of the basic amine by methods known to the art. Exemplaryof the acid addition salts that are included in this invention aremaleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate,benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide,sulfate, phosphate and nitrate salts.

As stated above, the compounds of the invention possess the ability toinhibit PAT or polyamine synthesis, properties that are exploited in thetreatment of any of a number of diseases or conditions, most notablycancer. A composition of this invention may be active per se, or may actas a “pro-drug” that is converted in vivo to active form.

The compounds of the invention, as well as the pharmaceuticallyacceptable salts thereof, may be incorporated into convenient dosageforms, such as capsules, impregnated wafers, tablets or injectablepreparations. Solid or liquid pharmaceutically acceptable carriers maybe employed.

Preferably, the compounds of the invention are administeredsystemically, e.g., by injection. When used, injection may be by anyknown route, preferably intravenous, subcutaneous, intramuscular,intracranial or intraperitoneal. Injectables can be prepared inconventional forms, either as solutions or suspensions, solid formssuitable for solution or suspension in liquid prior to injection, or asemulsions.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearateand stearic acid. Liquid carriers include syrup, peanut oil, olive oil,saline, water, dextrose, glycerol and the like. Similarly, the carrieror diluent may include any prolonged release material, such as glycerylmonostearate or glyceryl distearate, alone or with a wax. When a liquidcarrier is used, the preparation may be in the form of a syrup, elixir,emulsion, soft gelatin capsule, sterile injectable liquid (e.g., asolution), such as an ampoule, or an aqueous or nonaqueous liquidsuspension. A summary of such pharmaceutical compositions may be found,for example, in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa. (Gennaro 18th ed. 1990).

The pharmaceutical preparations are made following conventionaltechniques of pharmaceutical chemistry involving such steps as mixing,granulating and compressing, when necessary for tablet forms, or mixing,filling and dissolving the ingredients, as appropriate, to give thedesired products for oral or parenteral, including , topical,transdermal, intravaginal, intranasal, intrabronchial, intracranial,intraocular, intraaural and rectal administration. The pharmaceuticalcompositions may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand so forth.

Though the preferred routes of administration are systemic thepharmaceutical composition may be administered topically ortransdermally, e.g., as an ointment, cream or gel; orally; rectally;e.g., as a suppository, parenterally, by injection or continuously byinfusion; intravaginally; intranasally; intrabronchially; intracraniallyintra-aurally; or intraocularly.

For topical application, the compound may be incorporated into topicallyapplied vehicles such as a salve or ointment. The carrier for the activeingredient may be either in sprayable or nonsprayable form.Non-sprayable forms can be semi-solid or solid forms comprising acarrier indigenous to topical application and having a dynamic viscositypreferably greater than that of water. Suitable formulations include,but are not limited to, solution, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like. If desired, thesemay be sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers, or salts for influencing osmoticpressure and the like. Preferred vehicles for non-sprayable topicalpreparations include ointment bases, e.g., polyethylene glycol-1000(PEG-1000); conventional creams such as HEB cream; gels; as well aspetroleum jelly and the like.

Also suitable for topic application are sprayable aerosol preparationswherein the peptide, preferably in combination with a solid or liquidinert carrier material, is packaged in a squeeze bottle or in admixturewith a pressurized volatile, normally gaseous propellant. The aerosolpreparations can contain solvents, buffers, surfactants, perfumes,and/or antioxidants in addition to the compounds of the invention.

For the preferred topical applications, especially for humans, it ispreferred to administer an effective amount of the compound to anaffected area, e.g, skin surface, mucous membrane, eyes, etc. Thisamount will generally range from about 0.001 mg to about 1 g perapplication, depending upon the area to be treated, the severity of thesymptoms, and the nature of the topical vehicle employed.

The compositions of the invention be given in combination with one ormore additional compounds that are used to treat the disease orcondition. For treating cancer, the polyamine derivatives are given incombination with anti-tumor agents, such as mitotic inhibitors, e.g.,vinblastine; alkylating agents, e.g., cyclophosphamide; folateinhibitors, e.g., methotrexate, pritrexim or trimetrexate;antimetabolites, e.g, 5-fluorouracil and cytosine arabinoside;intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes orenzyme inhibitors, e.g., asparaginase; topoisomerase inhibitors, e.g.,etoposide; or biological response modifiers, e.g., interferon. In fact,pharmaceutical compositions comprising any known cancer therapeutic incombination with the substituted polyamines disclosed herein are withinthe scope of this invention. Most preferably, the present compounds areadministered in combination with a polyamine synthesis inhibitor such asDFMO.

The pharmaceutical compositions of the invention may also comprise oneor more other medicaments such as anti-infectives includingantibacterial, anti-fungal, anti-parasitic, anti-viral, andanti-coccidial agents.

Typical single dosages of the compounds of this invention are betweenabout 1 ng and about 10 g/kg body weight. The dose is preferably isbetween about 0.01 mg and about 1 g/kg body wt. and, most preferably,between about 0.1 mg and about 100 mg/kg body wt. For topicaladministration, dosages in the range of about 0.01-20% concentration ofthe compound, preferably 1-5%, are suggested. A total daily dosage inthe range of about 1-500 mg is preferred for oral administration. Theforegoing ranges are, however, suggestive, as the number of variables inregard to an individual treatment regime is large, and considerableexcursions from these recommended values are expected.

Effective amounts or doses of the compound for treating a disease orcondition can be determined using recognized in vitro systems or in vivoanimal models for the particular disease or condition. In the case ofcancer, many art-recognized models are known and are representative of abroad spectrum of human tumors. The compounds may be tested forinhibition of tumor cell growth in culture using standard assays withany of a multitude of tumor cell lines of human or nonhuman animalorigin. Many of these approaches, including animal models, are describedin detail in Geran, R. I. et al., “Protocols for Screening ChemicalAgents and Natural Products Against Animal Tumors and Other BiologicalSystems (Third Edition)”, Canc. Chemother. Reports, Part 3, 3:1-112,which is hereby incorporated by reference in its entirety.

Parallel Library Synthesis

Combinatorial Approaches to Polyamines and Analogues

Combinatorial chemistry, a rapidly changing field of molecularexploration, is still in its infancy. For reviews, see Lam, K. S.,Anticancer Drug Des. 12:145-167, 1997; Salemme, F. R. et al.; Structure5:319-324, 1997; Gordon, E. M. et al., J. Med Chem. 37:1385-1401, 1994;Gallop, M. A. et al., J. Med. Chem. 37:1233-1251, 1994). Thepharmaceutical industry, is now realizing that the original approach ofthe combined synthesis of hundreds to thousands of compounds in one“flask” followed by testing and deconvoluting the results is a tediousprocess with many pitfalls. The more traditional approach of medicinalchemistry, that is, the synthesis and testing of one compound at a time,yields more reliable and informative results about the SAR around atarget. The trend in combinatorial chemistry is therefore towardsynthesis of multiple compounds at once, with each in a separatecontainer. Therefore, many have adopted this one-compound/one-wellparallel synthetic approach. While many lead compounds have beengenerated this way, the chemistries do not necessarily lead to amolecule with the necessary drug-like characteristics.

The additional steps of lead optimization followed by incorporation ofdrug-like characteristics (molecular weight,hydrophilicity/hydrophobicity, transport, metabolism and stability)reduce drug development costs. Therefore it is preferred to pursue novelchemistries whereby the desired characteristics are incorporated intothe initial libraries. By decreasing the number of steps between leadidentification and production of a new drug candidate, costs can besignificantly reduced.

Using parallel synthesis starting with an array of sulfonyl chloridesand polyamines, a library of substituted polyamines can be synthesized.The parallel synthesis of 6 substituted polyamines starting from twodifferent amines and three different acid chlorides is described indetail in Example XV.

Using the appropriate available equipment as many as 24 compounds can besynthesized at the same time on one instrument. Commercially availableinstruments support high throughput parallel synthesis. Microtiter plateformats for synthesis of 96 compounds at a time are now routine, the 96multipin technology developed by Chiron Mimotopes PTY LTD (San Diego)being a typical example. This format is especially useful to synthesizepolyamine analogues with different length carbon linkers. Acombinatorial library synthesized with different polyamines and sulfonylchlorides is described in Example XVI.

Similarly, commercially available acid chlorides are used in parallelcombinatorial synthesis with different polyamines to yield a library ofmonosubstituted polyamine amides, as described in Example XVII.Substituted ureas are synthesized in parallel by activation of theappropriate (commercially available) amines with p-nitrophenylchloroformate. The product is then reacted with the commerciallyavailable amine to yield the desired substituted ureas as described inExample XVIII. Thousands of substituted carboxylic acids, sulfonicacids, sulfonyl chlorides, acid chlorides and amines are commerciallyavailable for coupling (directly or through a linker) to a variety ofcommercially available amines. Conventional methods can be used forderivatives that are not commercially available. Compounds comprisingthese libraries are evaluated in high-through-put screening assays fortheir potency in different disease contexts.

The chemistries described herein are readily applied in combinatorialsyntheses using the “one-compound/one-well approach.” Examples XVII andXVIII exemplify how this would be done. With this approach, many newanalogues are made in a short time so that the desired drug-likecharacteristics are quickly fashioned.

The present inventors have designed a method to synthesize novelcompounds for therapeutic uses and as probes for various assays. Suchcompounds are useful as drugs in a number of diseases, particularlyagainst cancer. They are also useful as a component in combination drugtherapy with, for example, a polyamine synthesis inhibitor such as DFMO(which inhibits omithine decarboxylase, ODC) or with other agents,thereby providing novel combination chemotherapy regimens. The compoundsof the present invention are also useful for treating other diseases orconditions in which polyamines play a role as described above.

Combinatorial Synthesis of Polyamine Analogues

This invention extends the repertoire of chemistries available toexplore SARs around drug targets for PAT inhibition and other relevantpharmacological and industrial targets. By combining the versatile andefficient NaBH₃CN reductive amination reagent with a soluble(MeO—PEG—OH) or insoluble polymeric support, a powerful combinatorialmethodology is provided. Additional important technical innovationsinclude the use of a Boc-like linker. that provides the desiredpolyamines as salts without the problem of additional residue remainingfrom the linker.

The direction of extension of the polyamine chain also ensures thatoveralkylation does not occur while at the same time ensuring highyields. Overalkylation results in di-alkyl side-products(MeO—PEGO-linker-CHO plus excess R-NH₂ instead of MeO—PEGO-linker-NH₂plus R—CHO).

Given the particularly mild nature of the chemistry involved, manyimportant and novel structural features can be readily incorporated intothe resulting polyamine products, including chiral substituents on thecarbon chain (using amino acid derived synthons), heterocyclicsubstituents, carbohydrates, nucleosides, conformational restraints and,finally, known drugs.

Synthetic Methods

The synthetic methods bring together the three major components of thisnovel technology in the synthetic cycle shown in FIG. 33. This includes(1) the use of soluble polymer to anchor the growing chain, (2)extenders and (3) terminators which block the chain extension.

First, an activated tert-alkoxycarbonyl MeO-PEG polymer is synthesizedas indicated in FIG. 34. Second, this polymer is reacted with a freeamino-protected aldehyde extender synthon. FIG. 35 depicts theproduction of these extenders from either commercially available aminoalcohols or the chiral amino acid precursor pool. Third, the syntheticcycle is followed using the reductive amination reaction with NaBH₃CNinitially to extend the backbone followed by an additional reductiveamination step with an aldehyde to terminate the secondary amineproduced above (FIG. 36). The final steps are the capping and acidcleavage from the polymeric support to provide the desired polyamineanalogue as an acid salt (FIG. 37).

1. Activated Polymer

The choice of MeO—PEG—OH (Catalog, Polyethylene Glycol Derivatives.Shearwater Polymers, Inc., 2307 Spring Branch Road, Huntsville, Ala.35801) as the polymeric support, is one of the key technical innovationsof this invention. This material has the unique properties of (a)solubility in most commonly used organic solvents (see Table 1; Bayer,E. et al., In Proc. Eur. Pept. Symp, 13th, 129, 1975), and (b)precipitability by the addition of diethyl ether. These propertiesovercome many of the problems of solid-phase synthesis. For example, itis well known that complete contact between reaction partners, without asolid-liquid phase barrier, greatly increases reaction rates and yields(Lloyd-Williams, P.; Tetrahedron, 49:11065-11133, 1993). As seen inTable 1 below, after completion of the reaction, the soluble polymericproduct can be precipitated from the reaction medium simply by pouringinto diethyl ether. In addition, other solid supports or chips can besubstituted.

TABLE 1 Solubility of MeO-PEG-OH 5000 at Room Temperature in WeightPercent Solvent Solubility (%) Water 55 CH₂Cl₂ 53 CHCl₃ 47 DMF 40Pyridine 40 CH₃OH 20 Benzene 10 Ethanol 60% 50 100% 0.1 100% at 34° C.20 Ethyl ether 0.01

Following filtration, the polymer can be further purified from excessreagents and side-products by recrystallization in absolute ethanol.These unique properties have been exploited by others for other types oflibraries. Janda et al. used this resin to produce pentapeptide andarylsulfonamide libraries (Han, M. et al., Proc. Natl. Acad Sci. USA,92:6419-6423, 1995; Janda, K. D. et al., Meth. Enzymol. 267:234-247,1996). Krepinsky and coworkers used the same selectively solublepolymeric support to produce structurally complex oligosaccharides(Douglas, S. P. et al., J. Am. Chem. Soc. 117:2116-2117, 1995;Krepinsky, J. J, US 5,616-698, 1997). These reports clearly show thefeasibility of this support in complicated and lengthy syntheticprocedures.

Hodges reported a novel procedure whereby an amine can be linked toMerrifield's polystyrene resin though a tert-alkoxycarbonylfunctionality (Hernandez, A. S. et al.; J. Org. Chem. 62:3153-3157,1997). This Boc-like linker has the distinct advantage of allowingcomplete removal of the linker moiety from the resin by acid treatmentin the final cleavage step. Therefore, application of this method in thepresent invention provides the final polyamine products as acid saltsfree from the linker residue.

Incorporation of this linker methodology with MeO—PEG—OH is depicted inFIG. 34. Formation of the lithium or sodium salt of MeO—PEG—OH 1a inTHF, followed by alkylation with 2-methyl-4-halo (or tosyl)-1-butene,will give the desired alkene ether 2a of the polymer. See (Douglas etal., supra; and U.S. Pat. No. 5,252,714) for examples. Oxymercurationwith Hg(OAc)₂, followed by reductive demetallation, gives the desiredtertiary alcohol 3 (Brown, H. C. et al., J. Org. Chem. 35:1844-1850,1970). The “one-pot” method of Hodges (supra) is then used to activatethis resin for reaction with the first amine subunit. Reaction withN,N′-carbonyldiimidazole and 4-N,N-dimethylaminopyridine gives theintermediate (tert-alkoxycarbonyl)imidazole that is activated bymethylation with methyl trifluoromethanesulfonate (MeOTf). The excessMeOTf is removed by treatment with Et₃N which itself does not react withthe activated imidazolide 4a.

2. Extender Units

Great flexibility in this technology lies in the choice of startingmaterials for extender synthesis. As shown in FIG. 35, by selection fromthe chiral amino acid pool, free amino/protected aldehydes are producedin high yield and purity in several easy steps. Unprotected amino acids5a can be directly reduced to their amino alcohols by treatment withNaBH₄ and I₂ (Bhaskarkanth, J. V. et al., J. Org. Chem. 56: 5964-5965,1991). It is anticipated that protection of the amino acid maynevertheless be necessary for the first, carboxylate reduction step,especially with the more complex amino acids. Selective protection ofthe more nucleophilic amino functionality with a trifluoroacetyl group(using S-ethyl trifluorothioacetate) yields the desired protected aminoalcohol 6a (Schallenberg, E. E., Calvin, M., J. Am. Chem. Soc.77:2779-2783, 1955). This protecting group is selected mainly because ofthe ease of introducing it and cleaving it with mild base.Pfitzner/Moffatt oxidation using dicyclocarbodiimide and dichloroaceticacid in DMSO followed by in situ aldehyde protection as a1,3-diphenylimidazolidine yields the crystalline fully protectedintermediate 7 (Pfitzner, K. E. et al., J. Am. Chem. Soc. 87:5661, 1965;Ranganathan, R. S. et al., J. Org. Chem. 39, 290, 1974).

Use of this aldehydic protecting group overcomes several problems atonce, for example, the often problematic purification of free aldehydes.Many of these 1,3-diphenylimidazolidine 7 compounds are very crystallineand easy to purify (Wanzlick, H. W. et al., Chem. Ber. 86:1463, 1953).Simple hydrolysis with aqueous ammonium hydroxide produces the chiralfree amine/protected aldehyde extender unit 8.

Commercially available amino alcohols 6a are being used in the sequenceof steps in FIG. 35. The amino protection, alcohol oxidation, aldehydeprotection and amide hydrolysis steps have been successful using 3, 4and 5-carbon chain amino alcohols 6a. These synthons 8a enableproduction of polyamine scaffolds mimicking the naturally occurringputrescine, spermidine and spermine.

3. Synthetic Cycle

With both the activated polymer 4a (FIG. 34) and the extender units 8a(FIG. 35) in hand, the coupling method is ready to be tested and used.The first extender unit 8a is reacted with the activated polymer to givethe protected aldehyde linked through a carbamate, 9a (FIG. 36).Selective deprotection of the 1,3-diphenylimidazolidine group from theterminal aldehyde of 9a produces the required starting substrate for thecoupling reaction. This hydrolysis, using a weak acid such as aceticacid, poses no difficulties since the Boc-like linker moiety is expectedto be cleaved under more strenuous acid conditions such as 10% TFA inCH₂CH₂. (Moffatt cleaved the 1,3-diphenylimidazolidine of 5′-aldehydicadenosine with Dowex 50 cation exchange resin (H⁺ form) in the presenceof a isopropylidene ketal (a more acid-sensitive group than is a Bocgroup); (Ranganathan et al., supra).

To optimize yields, other aldehydic protecting groups including acyclicacetals, cyclic acetals or the acid stable dithio acetals may beincorporated into the scheme. The polymeric aldehyde 10a is then reactedwith an excess of the next free amine/protected aldehyde subunit 8a(FIG. 35) under the Borch reductive amination conditions in a suitablesolvent such as THF or methanol (Borch, R. F. et al., J. Am. Chem. Soc.93:2897-2904, 1971). Reductive amination reactions on solid supports arewell-known (Sasaki, Y. et al. J. Med. Chem. 30:1162-1166, 1987; Gordon,D. W. et al., Bioorg. Med. Chem. Lett. 5:47-50, 1995; Devraj, R. et al.,J. Org. Chem. 61,:9368-9373, 1996).

Initial imine formation, catalyzed by trace AcOH, followed by additionof NaBH₃CN yields the methylene secondary amino product 11a (FIG. 36).The secondary amino function in 11a cannot react further with the excessamine used in the initial imine formation. By using an excess of thefree amino extender unit 8a, complete reaction is ensured. Completereaction is important in any multi-step solid phase synthetic methodsince lower reaction yields can potentially give complex productmixtures full of undesired species.

By performing the coupling in this synthetic direction with an excess ofthe amino extender, greater than 98% coupling yields are achieved Anexcess of the amine greatly increased the yield of the desired secondaryamine, based on the aldehyde component. It is expected that differentamino/aldehyde reaction partners will react at differing rates. However,by addition of excess amine and by allowing the reaction to proceed forlonger times, complete reaction is expected in all cases.

A second reductive amination reaction can now be performed on theresulting, free, secondary amino function of 11a. A wide assortment ofcommercially available aldehydes are suitable for this reaction, whichcan “dress” the resulting polyamine scaffold in interesting and novelways.

A large series of simple straight chain or branched alkyl aldehydes isavailable. These modified polyamines would have greater lipophilicproperties, thus increasing their ability to cross biological membranesand the blood-brain barrier. Any of a large number of unsaturated alkenealdehydes could also be used. A particularly interesting example isacrolein, that can serve as a protecting group thereby allowing returnto the secondary amine by deprotection of the resulting allyl amine withRh(Ph₃P)₃Cl catalyst (Laguzza, B. C. et al., Tetrahed. Lett.22:1483-1486, 1981). An alternative method for secondary amineproduction is the use of the tBoc group which is cleaved along with thelinker in the final reaction.

A wide variety of aromatic aldehydes is also available. Any of a seriesof substituted benzaldehyde derivatives together with a variety ofheterocyclic aromatic aldehydes could be used.

This potential can also be extended to more water soluble derivatives byusing carbohydrates and carbohydrate phosphates. With the versatilityand mild nature of the coupling chemistry, very complex molecules suchas nucleoside aldehydes could also be used to modify these newanalogues. The well-known highly avid binding of polyamines to thepolyphosphate backbone of DNA and RNA polymers creates the potential forspecific, tightly bound polycationic DNA “triple helix”analogues(Dempcy, R. D. et al., Proc. Natl. Acad. Sci. U.S.A.,93:4326-4330, 1996; Goodnow, Jr., R. A. et al., Tetrahedron Lett.38:3195-3198, and 3199-3202, 1997). Such a series would allow analysisof the molecular space surrounding the polyamine targets (describedbelow). With great synthetic flexibility not only in the polyaminescaffold (obtained through choice of the extender units), but also inthe range of terminators available to dress the resulting scaffold, itis possible to create great molecular diversity around polyamineanalogues.

With the fully terminated and “modified” polyamine in hand, the finalcapping and cleavage from the polymeric support are performed (FIG. 37).The chemistry of the final capping is again very versatile. By milddeprotection of the 1,3-diphenylimidazolidine 12a (FIG. 36) to produce10a (FIGS. 36 and 37), and reaction with ammonia under reductiveamination conditions, the cleaved polyamine analogue 15a containsprimary amino functions at both ends. To create a structure with asecondary amino function at one end, a reaction of the free terminalaldehyde with a primary amine is performed. Reaction with a secondaryamine would yield a tertiary amine at the terminal position. Another wayof gaining a bifunctional terminal, tertiary amine is by reaction with aprimary amine followed by reaction with an aldehydic terminator asabove.

Finally, selective modification of either end of the polyamine isachieved by capping the terminus, cleaving from the polymeric supportand reacting with an aldehyde. Because this will result in polyaminesthat are selectively protected at all amino functions from furtherreaction under reductive amination conditions, the synthetic variabilityof this approach is virtually unlimited.

4. Analytical and Purification Procedures

Several powerful analytical techniques may be used to follow each of thetypes of reactions described above. Throughout the synthesis, anyintermediate can be analyzed by ¹H- and ¹³C-NMR techniques (Han et al.,supra; Janda et al, supra; Douglas et al., supra; Krepinsky et al.,supra). The degree of functionalization of the MeO—PEG—OH resin can beanalyzed by reaction with phenylisocyanate and UV analysis of theresulting carbamate groups that form on the unreacted polymeric hydroxylgroups. (Analysis of unreacted MeO—PEG—OH (M.W.=5000 daltons) gives anε₂₃₆ value of 17,500 M⁻¹ cm⁻¹). Free aldehyde groups are quantified byreaction with 2,4-dinitrophenylhydrazine followed by UV quantification.Free amino groups are quantified by reaction with ninhydrin followed byUV analysis (Kaiser, E. et al., Anal. Biochem. 34:595, 1979). Infraredspectroscopy is used for functional group identification. Using theinformation from these techniques together with weights of the finalproducts after cleavage from the polymer, correlations between theindividual analytical methods are made. The final, cleaved products arethoroughly analyzed by standard techniques including: ¹H- and ¹³C-NMR,UV analysis where applicable, IR spectra, melting points, HPLC and TLCretention times and elemental analysis. If these techniques show thatfurther purification is warranted before biological testing,chromatographic techniques such as cation exchange or reverse-phasechromatography are effectively employed for that purpose (Siegel, M. G.et al., Tetrahedron Lett. 38:3557-3360, 1997). Examples of compoundssynthesized by this approach are shown in FIG. 35.

Solid-Phase Synthesis

The approach outlined above for the liquid-phase synthesis can also beperformed using as solid-phase supports polystyrene resins, chip-basedsystems, multi-pin systems and microwells containing hydroxyl groups.Many solid supports with a hydroxyl linker are available, e.g., the Wangresin (Wang, S.-W., J. Am. Chem. Soc., 95:1128-1333, 1973). Many linkershave been described, and major efforts are under way to design“linker-less” resins that, after cleavage, eliminate the linker to yieldthe compound of choice.

In contrast to the liquid-based approach, reaction conditions in thesolid-phase approach are designed to give optimal yields and minimizeside-reactions or incomplete reactions. Therefore, excess reagents areused and are washed away; unwanted reagents are also removable withscavenger resins (Booth R. J. et al., J. Am. Chem. Soc. 119: 4882,1997). Catalytic resins can be used to speed up reactions.

An example of a solid support with alternative linking group, tosynthesize a polyamine analogue is shown in FIG. 39, illustrating3,4-dihydro-2H-pyran-2-yl-methoxymethyl polystyrene 16a(Calbiochem/Novabiochem, La Jolla, Calif.) (Thompson, L. A. et al.,Tetrahed Lett., 35: 9333, 1994). Reaction of 12 with the blockedaminoaldehyde yields 17a which is deblocked with HgCl₂/HgO to aldehyde18a. Reaction of aldehyde 18a with a blocked aminoaldehyde in thepresence of NaBH₃CN yield 19a with an extended chain containing asecondary amine which blocked with a terminator aldehyde to yield 19a.Compound 19a can either be deblocked and cleaved from the resin to yielda product or can be reacted in a next cycle. The desired productcontaining a hydroxy-tail is released from the resin by treatment with95% TFA/5% H₂O.

The multipin-method is based on a modular 8×12 matrix of dimensionallystable polypropylene/polyethylene pins to which a graft polymer iscovalently linked. Synthesis is performed upon the graft polymer, whichcan be varied to suit the application. Examples of multipin systems(Chiron Mimotopes, San Diego, Calif.) containing different linker groupsare shown in FIG. 40. The Rink amide linker is shown as structure 23acoupled to the pin P (Rink H., Tetrahed. Lett., 28:1787-1790, 1987).This linker requires the removal of the Fmoc protecting group prior touse. The desired product is then synthesized by building it out from thefree amino group. After completion of the synthesis the product can becleaved using 5% TFA/CH₂Cl₂ to give primary amides. This group is stableto weak acid and base. Pins with structures 23a and 25a are usedprimarily for coupling carboxylic acids. The stability of 23a s good,and it is only labile to strong base and is cleaved with 95% TFA/H₂O. Incontrast 25a is stable to strong base and is cleaved with 95% TFA/H₂O(Valerio, R. M. et al., Int. J. Peptide Protein Res. 44:158-165, 1994).Structure 24a, is used to couple acids which can be cleaved with eitherNaOH or NH₂R to give amides. This pin type can be used in the presenceof HF, TFA and weak base. Structure 27a performs similarly to 25a, butis more suitable for milder acids and is generally more labile (Bray, A.M. et al., J. Org. Chem. 59:2197-2203, 1994). Structure 26a is used tocouple carboxylic acid, is cleaved following alkylation with CH₂N₂ withNaOH or NH₂R (to give amides) and is stable to strong acid and strongbases prior to alkylation. It will be evident to those skilled in theart that these solid supports can be incorporated in the approachesdescribed above. In addition, other solid supports known in the art canbe combined with known chemistries to generate polyamine analoguescontaining different functional groups determined by the particularcleavable linker used.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE I Synthesis of N¹-dansylspermine 3

Synthesis of N¹-dansylspermine is illustrated in FIG. 7. To 0.81 g (4mmole) of spermine and 0.1 g (mmole) of triethylamine in 30 ml dryCH₂Cl₂ cooled down to 4C, was added dropwise 0.27 g (1 mmole) dansylchloride dissolved in 20 ml dry CH₂Cl₂ over 90 minutes. The temperaturewas allowed to rise to ambient temperature and was stirred for 16 hourswhen it was filtered to remove triethylamine hydrochloride. Theprecipitate was washed with 25 ml CH₂Cl₂ and the combined CH₂Cl₂extracts was extracted with 2×25 ml 5% Na₂CO₃ and 1×25 ml water. TheCH₂Cl₂ was filtered through Whatman no 1 filter paper and evaporated todryness to yield 0.45 g. Thin layer chromatography on silica gel inisopropanol:pyridine:acetic acid:water (4:1:1:2) showed no startingspermine and mainly two spots, when sprayed with 0.2% ninhydrin/ethanol.The material was dissolved in 8 ml 1.0 M ammonium acetate pH 7.4 and waschromatographed on a Biorad 70 weak cation exchanger (1.5×48 cm) using apH gradient between 1.0 M ammonium acetate and 1.25 M hydrochloric acidover 500 ml with a flow rate of 0.5 ml per minute, collecting 8 mlfractions. Fractions containing a single spot were collected, adjustedto pH 10.5 and extracted with 2×25 ml CH₂Cl₂. This CH₂Cl₂ fraction wasfiltered through Whatman filter paper and evaporated to dryness. Thesolid product was dissolved in ethanol acidified with hydrochloric acidand recrystallized from ethanol to yield 0.14 g of N¹-dansylspermine(also termed monodansylspermine or “MDS”). The NMR spectrum confirmedthe structure. The products can be purified by recrystallization without any ion exchange chromatography.

EXAMPLE II Synthesis of N¹-(1-pyrenylsulfonyl)spermine 15

Synthesis of N¹-(1-pyrenylsulfonyl)spermine) is illustrated in FIG. 5.To 0.56 g (2.8 mmole) of spermine and 0.069 g (0.69 mmole) oftriethylamine in 25 ml dry CH₂Cl₂ cooled down to 4° C., was addeddrop-wise 0.20 g (0.69 mmole) dansyl chloride 1-pyrenesulfonyl chloridedissolved in 20 ml dry CH₂Cl₂ over 30 minutes. The temperature wasallowed to rise to ambient temperature and was stirred for 16 hours whenit was filtered to remove triethylamine hydrochloride.

The precipitate was washed with 25 ml CH₂Cl₂ and the combined CH₂Cl₂extracts were evaporated to dryness and dissolved in ethyl acetate whichwas extracted with twice with 25 ml 5% Na₂CO₃ and once with 25 ml water.The ethyl acetate was filtered through Whatman no I filter paper andevaporated to dryness to yield 0.26 g.

Thin layer chromatography on silica gel inisopropanol:pyridine:acetic-acid:water (4:1:1:2) showed no startingspermine and mainly two spots, when sprayed with 0.2% ninhydrin/ethanol.

The material was dissolved in 8 ml 1.0 M ammonium acetate pH 7.4/MeOH1:1 and was chromatographed on a Biorad 70 weak cation exchanger (1.5×48cm) using a pH gradient between 1.0 M ammonium acetate pH 7.4 and 1.25 Mhydrochloric acid/methanol (1:1) over 500 ml with a flow rate of 0.5 mlper minute, collecting 8 ml fractions. Fractions containing a singlespot were collected, adjusted to pH 10.5 and extracted with 2×25 mlethyl acetate. This ethyl acetate fraction was filtered through Whatmanfilter paper and evaporated to dryness. The solid product was dissolvedin ethanol acidified with hydrochloric acid and recrystallized fromethanol to yield 0.10 g of N¹-(1-pyrenylsulfonyl)spermine·3HCl. TLCindicated a single component and NMR spectrum confirmed the structure.

EXAMPLE III Synthesis of N¹-((1-carbonyl)-4-(1-pyrenyl)butane)spermine37

Synthesis of N¹-((1-carbonyl)4-(1-pyrenyl)butane)spermine is illustratedin FIG. 6. To 0.29 g (1 mmole) of 1-pyrenebutyric acid dissolved inCHCl₃ with heating were added 0.19 g (1 mmole) of EDC 0.12 g (1 mmole)of N-hydroxysuccinamide and was stirred at room temperature for 30minutes when this solution was added drop-wise to 0.82 g (4 mmole)spermine dissolved in 20 ml CHCl₃. The reaction was allowed to proceedfor another 4 hours when it was diluted with an equal volume ofethylacetate. This solution was extracted with 25 ml 5% Na₂CO₃ and oncewith 25 ml water. The organic solution was filtered through Whatman no 1filter paper and evaporated to dryness to yield 0.25 g.

Thin layer chromatography on silica gel in isopropanol:pyridine:aceticacid:water (4:1:1:2) showed no starting spermine and mainly two spots,when sprayed with 0.2% ninhydrin/ethanol.

The material was dissolved in 8 ml 1.0 M ammonium acetate pH7.4/methanol 1:1 and was chromatographed on a Biorad 70 weak cationexchanger (1.5×48 cm) using a pH gradient between 1.0 M ammonium acetateand 1.25 M hydrochloric acid/methanol (1:1) over 500 ml with a flow rateof 0.5 ml per minute, collecting 8 ml fractions. Fractions containing asingle spot were collected, adjusted to pH 10.5 and extracted with 2×25ml ethyl acetate. This ethyl acetate fraction was filtered throughWhatman filter paper and evaporated to dryness. The solid product wasdissolved in ethanol acidified with hydrochloric acid and recrystallizedfrom ethanol to yield 0.13 g ofN¹-((1-carbonyl)-4-(1-pyrenyl)butane)spermine. TLC indicated a singlecomponent and NMR spectrum confirmed the structure.

EXAMPLE IV N-(1-anthracenyl)-N′-(N1-spermidyl)urea (9)

Synthesis of N-(1-anthracenyl)-N′-(N1-spermidyl)urea is illustrated inFIG. 4. A solution of 1 g of 1-aminoanthracene (5.2 mmole) and 1.04 gp-nitrophenyl chloroformate (5.2 mmole) in 100 ml benzene was refluxedusing an air condenser until no more HCl gas escaped as measured with pHpaper (3 hours). The desired product,N-(1-anthracenyl)-O-(p-nitrophenyl)urea (1.6 g; 86% yield) was filteredfrom the cooled reaction and washed with benzene. This product was usedwithout further purification.

To 0.5 g (2.5 mmole) spermine in 30 ml dichloromethane was addeddropwise 0.18 g (0.5 mmole) of the urethane in 20 ml dichloromethane.The reaction was allowed to proceed for 16 hours when it was extracted2×50 ml 5% Na₂CO₃ solution followed by 1×50 ml water. The filteredsolution was evaporated to dryness on a high vacuum. The residue wasdissolved in MeOH and acidified with 4 equivalents of 6N HCl acidsolution. This solution was evaporated to dryness and was thenrecrystallized from EtOH/MeOH to yield 27.5 mg of compound that showedmainly one spot on silica gel TLC (isopropanol:pyridine:aceticacid:water; 4:1:1:2).

EXAMPLE V Synthesis of N-(N¹-spermidyl)-2-(naphthyl)acetamide (103)

A synthetic scheme as described in Example IX ??? is carried out, withthe difference that the starting compound is 1-naphthylacetic anhydride(instead of N6-(dansyl)-6-aminocaproyl-N-hydroxysuccinimide ester). Thisyields the product N-(N¹-spermidyl)-2-(naphthyl)acetamide as shownbelow:

Using known chemistries the chain length can be increased as desired. Apreferred length is n=1 to 10.

EXAMPLE VI N-(N¹-spermidyl)-2-(naphthoxy)acetamide (104)

The same synthetic is carried out using as starting material(2-naphthoxy)-acetic acid, N-hydroxysuccinimide ester, so that theproduct is N-(N¹-spermidyl)-2-(naphthoxy)acetamide as shown below:

Using known chemistries the chain length can be increased as desired. Apreferred length is n=1 to 10.

EXAMPLE VII Synthesis of O-(Fluorenylmethyl)-N-(N1-spermidyl)urethane

A synthetic scheme as described in Example II is carried out usingstarting compound 9, fluorenylmethyl chloroformate instead of1-pyrenylsulfonyl chloride as shown below.

EXAMPLE VIII

Disubstituted functionalizable compounds are well known in the art, forexample sulfonyl chlorides, benzoyl chlorides, cyanates, thiocyanates,etc. The reaction of 2,6-naphthalene disulfonyl chloride with spermineis shown below.

EXAMPLE IX N¹-[(N⁶-dansyl)-6-aminocaproyl]spermine (DACS 4) by Method 1

Synthesis of DACS by method 1 is illustrated in FIG. 8. The reactantsand product are shown below. To 0.55 g spermine (2.7 mmole ) in 20 mldichloromethane cooled in an ice bath was added drop-wise 0.125 g ofN6-(dansyl)-6-aminocaproyl-N-hydroxysuccinimide ester (0.27 mmole)dissolved in 10 ml dichloromethane over 30 minutes. The reaction wasstirred for 16 hours at ambient temperature when it was filtered toremove precipitate. The filtrate was diluted with 30 ml CHC₃ and wasextracted 2×50 ml 5% Na₂CO₃ solution followed by 1×50 ml distilledwater. The organic phase was filtered and evaporated to dryness. Theresidue (0.20 g) was dissolved in 7 ml methanol and acidified with 5equivalents of 6N HCl. The solvent was evaporated and the solid wasrecrystallized from ethanol/methanol gave 0.073 g (39% yield ) of thedesired product. Silica gel TLC in isopropanol:pyridine:aceticacid:water (4:1:1:2) showed a single fluorescent spot which also gave aninhydrin positive spot. Nominal mass spectrometry, ion pair reversedphase chromatography and NMR confirmed the identity and purity of thecompound.

EXAMPLE X 4-Nitrophenyl 6-(N-(t-butoxycarbonyl)amino)hexonate 108

This compound, illustrated as an intermediate to DACS is shown in FIG.9. To a dry round-bottom flask was added 11.55 g (50 mmol) of6-(N-(t-butoxycarbonyl)amino)hexanoic acid 1 (available fromNovoCalbiochem), 12.4 g (60 mmol) of dicyclclohexylcarbodiimide and 8.35g (60 mmol) of 4-nitrophenol. To these solids was added 150 mL of dryEtOAc under argon at r.t. to produce an off-white heterogeneoussuspension. After 3 h at r.t. the solid DCU was filtered off through apad of Celite and this pad was washed 3× with 50 mL of EtOAc. Thecombined filtrates were evaporated to give 27 g yellow solid. This wascrystallized from 200 mL of abs. EtOH to give 13.54 g (77%) white solidas first crop. TLC (silica gel, CHCl₃) Rf 0.7. NMR confirmed theidentity of the compound.

EXAMPLE XI 4-Nitrophenyl 6-aminohexonate trifluoroacetate Salt 109

This compound, illustrated as an intermediate to DACS is shown in FIG.9. To a solution of 5.0 g (14.2 mmol) of 108 in 30 mL of CH₂Cl₂ wasadded 15 mL of trifluoroacetic acid at r.t. Many bubbles formed in theclear reaction solution. After 1 h the solvents were removed underreduced pressure to give a clear oil. This oil was triturated withdiethyl ether to form a white waxy solid which was dried under highvacuum. TLC (Rf 0.05 in 10% MeOH in CHCl₃) showed the product was pureenough for the next step. Yield 5.25 g white solid (100%).

EXAMPLE XII 4-Nitrophenyl 6-(N-(dansyl)amino)hexonate 110

This compound, illustrated as an intermediate to DACS is shown in FIG.9. To the suspension of 4.2 g (11.5 mmol) of 109 in 50 mL of dry CH₂Cl₂was added 3.71 g (13.8 mmol) of dansyl chloride as a solid, followed by4.8 mL (34.5 mmol) of dry Et3N dropwise through a syringe under argon atr.t. The resulting yellow solution was stirred at r.t. for 18 hr. whenthe solvents were evaporated to give a green oily solid. This materialwas dissolved in 250 mL of CHCl₃ and washed with 100 mL of 0.1 N HCl,H₂O then brine. The organic layer was dried and evaporated to give 5.85g green oily solid. This was crystallized from 100 mL of abs. EtOH togive 2.136 g (38%) yellow solid from the first crop. The mother liquorcan be crystallized for a second crop or purified by columnchromatography on silica gel using CHCl₃ then 10% EtOAc in CHCl₃ foradditional pure product. M.p. 84-86C. NMR confirmed the identity of thecompound.

EXAMPLE XIII N¹-[(N⁶-dansyl)-6-aminocaproyl]spermine (DACS 4) by Method2

This synthetic method is illustrated in FIG. 9. To a clear solution of72.8 mg (0.36 mmole) of spermine in 2 mL of MeOH is added 2.0 mL of 0.15M MeOH solution (0.30 mmol) of 110 dropwise at r.t. After 1 drop wasadded a very bright yellow color appeared. This yellow solution wasstirred for 15 min. when the solvent was evaporated to give 220 mg of ayellow, oily solid. The crude product was dissolved in 1.0 mL of 0.5 MHCl and applied to a 1×36 cm column of C-18 RP silica gel (Bakerbond#7025-01) in 20/80 MeOH:0.5 M HCl. Elution with the same solvent gave 79mg (38%) pure hydrochloride salt as a white solid. TLC using 4/1/1/2isopropanol:acetic acid:pyridine:H₂O gives an Rf of 0.70 for DACS, 0.90for diacyl side product and 0.18 for spermine. NMR confirmed theidentity of the compound.

EXAMPLE XIV N¹-[6-aminocaproylspermine] 171

This reaction scheme is carried out as described in detail below.

To a clear solution of 125 mg (0.62 mmol) of spermine in 5.0 mL of MeOHwas added a suspension of 181 mg (0.52 mmol) of 3 in 5.0 mL of MeOH. Theresulting bright yellow solution was stirred at r.t. for 15 min. whenthe solvents were evaporated. The resulting yellow solid was dissolvedin 10 mL of H₂O and applied to 1×30 cm column of BioRex 70 (NH₄ ⁺ form)resin. Elution was performed by a linear gradient of 0 to 1 N NH4OH. Theproduct containing fractions were evaporated to give 181 mg of N-t-Bocintermediate that was contaminated with 4-nitrophenol. This material wasdissolved in 3.0 mL of H₂O and 3.0 mL of 6 N HCl was added at r.t. After2 h at r.t. the clear solution was extracted 3× with 5 mL of CHCl₃, 1×with EtOAc then 1× with CHCl₃ again. The aqueous layer was thenevaporated to give 220 mg (92%) white solid. NMR confirmed the identityof the compound.

EXAMPLE XV Parallel Combinatorial Library Synthesis

The general reaction involved in the parallel synthesis is shown in thereaction below:

In each of three 10 ml reaction vials (React-Vial™ Pierce, Rockford,Ill.) were placed 0.74 mmol of spermine and 0.15 mmol of triethylamine.Similarly in three additional reaction vials were placed 0.74 mmol ofspermine and 0.15 mmol of triethylamine. Similarly in three additionalreaction vials were also placed 0.74 mmol of putrescine and 0.15 mmoltriethylamine. To each of these flask were added 2.5 ml dry CH₂Cl₂ andthe flasks were closed with a septum and cooled down to −20° C. in aReact-block™ aluminum block for 45 minutes, when it was placed in aReacti-Therm™ Heating/Stirring Module, with heating switched off. Threeacid chlorides (1-naphthylsulfonyl chloride, 2-naphthylsulfonyl chlorideand 10-carnphorsulfonyl chloride) in 2.5 ml CH₂Cl₂ were added dropwiseover 15 minutes via a 2.5 ml syringe (All-PP/PE, Aldrich, Milwaukee,Wis.) through the septum to each of spermine and putrescine. Each vialcontained also an exhaust consisting of a 2.5 ml syringe filled withanhydrous CaCl₂ with out the plunger. The reactions were allowed toproceed for 16 hours at ambient temperature when it was extracted 2×2.5ml 5% sodium carbonate solution followed by 2×2.5 ml water. To theorganic solvents were added 2.5 ml methanol and 5 equivalents of a 6NHCl solution. The solvent was evaporated with argon and dried on a highvacuum. Silica gel TLC with isopropanol:acetic acid:pyridine:water4:1:1:2 showed mainly one component with either UV/fluorescence or 0.2%ninhydrin in ethanol staining. Purity was estimated as to be greaterthan 80%. The structures, yield and inhibition of the polyaminetransporter is shown in Table 1, below.

EXAMPLE XVI Parallel Library Synthesis (a)

Using the Reacti-Therm™Heating/Stirring Module triple module, twentyfour 10 ml vials are used at the same time, thereby increasingsubstantially the number of compounds that can be synthesized inparallel. In addition more than one of these modules can be used at thesame time. Using this approach with the commercially available amineslisted below and other amines synthesized as described above, librariesof compounds are synthesized with commercially available sulfonylchlorides (from Aldrich Chemical Company, Maybridge Chemical Company,Ryan Scientific Inc., to name a few) in a manner as described in ExampleI.

List of Polyamines: N-(3-aminopropyl)-1,3-propanediamine, N,N′-bis-(3-aminopropyl)ethylenediamine N,N′-bis(3-aminopropyl)piperazineN,N′-bis(3-aminopropyl)-1,3- propanediamine N,N′-bis(2-aminoethyl)-1,3-Tris(3-aminopropyl)amine propanediamine Tris(2-aminoethyl)amine

TABLE 1 Structures, Yield and Inhibition of the Polyamine Transporter inMDA-MB-231 Cell Line Compound % Yield Ki μM

94.6 0.19

84.8 >30

82.6 0.15

88.8 5

59.6 >10

79.9 >30

EXAMPLE XVII Parallel Library Synthesis (b)

A library is synthesized as in Example I, with carboxylic halides in theplace of the sulfonyl chlorides, as indicated below. Useful carboxylichalides are commercially available from varies source.

EXAMPLE XVIII Synthesis of Library of N′-“headgroup”-N″-(N1-spermidyl)urea

A synthesis of the type shown in Example IV is carried out, with thedifference that the starting urethanes are first synthesized in parallelusing different aromatic amines as processors.

EXAMPLE XIX Cell Growth and its Inhibition by Polyamine Analogues

The present investors have developed a growth assay to use in screeningfor transport inhibitors that are synergistic with ODC inhibitors. Theestrogen insensitive human breast carcinoma MDA-MB-231 cell line as theprimary cell line in the screen. This cell line, as with many breastcancers, has a high rate of polyamine transport (Anticancer Res. (1991)11:1807-1814). In order to optimize the screen for polyamine transportinhibition, 1.0 μM spermidine was added to media to reverse the effectsof ODC inhibitors. The assay was also performed over seven days becausethis allows for the greatest dynamic range in cell growth due to themechanism of ODC inhibitors. Cells need to divide several times beforethe intracellular level of polyamines begin to decrease to growthinhibitory levels. Therefore, growth does not significantly cease untilthe third to fourth day.

When used to screen for polyamine transport inhibitors, the growth assayalone does not verify a reduction of polyamine uptake. Therefore, thegrowth assay and a kinetic transport assay have been used to validatetransport inhibition.

A. DACS Inhibits Polyamine Transport and Acts Synergistically with ODCInhibitors

Screening of thousands of compounds has permitted the present inventorsand their colleagues to identify a transport inhibitor that inhibitsspermidine uptake with a K_(i) of 8 nM, putrescine uptake with a K_(i)of 5.4 nM and has an IC₅₀ of 0.6 μM for growth in combination with anODC inhibitor (FIG. 22). Over 100 analogues of this compound have beensynthesized and SAR data has been accumulating around the structuralfeatures necessary to inhibit polyamine uptake. Additional compoundshave been discovered with even greater potency than DACS, but not asexhaustively studied as described below. Under the assay conditionsdescribed above, with 1.0 μM supplemented polyamines, there is no growthreduction due to ODC inhibition alone. In addition, DACS is not growthinhibitory alone until very high concentrations (300 μM) are reached.DACS makes the previously ineffective ODC inhibitors very effective asgrowth inhibitors in the presence of polyamines.

Growth inhibition by the combination of DACS and an ODC inhibitor in thepresence of polyamines (FIG. 23) mimics the effects of the ODC inhibitorin the absence of significant extracellular polyamines. Growthinhibition began to appear at day 2 and cell growth was reduced 69% byday 3. Growth eventually reached a plateau with the ODC inhibitorcombined with DACS but continued in the absence of DACS. This effectappears to be cytostatic in this cell line but, for prolonged periods oftime, may be cytotoxic.

B. DACS is Effective in the Presence of Natural Polyamines

Extracellular spermidine, spermine and putrescine can reverse theeffects of ODC inhibitors through increased uptake into the cell. Themajor excreted forms of polyamines (N¹-acetylspermine andN¹-acetylspermidine) can also reverse the effect of ODC inhibitors. DACSprevents the natural polyamines, putrescine, spermidine,N¹-acetylspermine and N¹-acetylspermidine, from rescuing the cells fromODC inhibition. This is significant for several reasons. Reports in theliterature suggest that there are more than one transporter. If this istrue, DACS is effective at blocking the uptake of all of the polyaminesat low concentrations.

C. DACS is Effective Against Several Types of Cancers

DACS was tested in vitro in combination with ODC inhibitors againstseveral human cancer cell lines. These include T-cell acutelymphoblastic leukemias (ALL), glioblastomas, prostate, and colon celllines. DACS is effective against all these tumor cell lines in vitro.FIG. 24 shows the effects of DACS on PC-3 prostate cancer cells.

EXAMPLE XX Screening of Polyamine Analogues in Transport and GrowthAssays

The effect of a number of potential PAT transport inhibitors on PAT andgrowth of MDA cells is summarized in FIG. 2 (3-98). The ratio, R is theIC₅₀ for polyamine alone relative to the IC₅₀ for the polyamine analoguecombined with an ODC inhibitor. This value of R, indicates the relativelevel of “synergism” between the polyamine analogue and ODC inhibitor.Under the growth assay conditions, the ODC inhibitor alone shows noinhibition.

EXAMPLE XXI Transport Inhibitors Inhibit Polyamine-Utilizing Enzymes

A study was conducted to determine whether the compositions of thepresent invention, designed as PAT inhibitors, had other activities onthe PA system. Specifically, the ability of DACS to inhibit an enzymeinvolved in PA recycling was evaluated. The method used was as describedin Casero, R. A. et al., Biochem. J270:615-620 (1990) herebyincorporated by reference in its entirety. This assay measures theincorporation of ¹⁴C-labeled acetyl CoA into spermidine to formacetylspermidine. Varying concentrations of DACS were added to areaction mixture containing HEPES buffer, pH 7.8, 1 mM spermidine, and 1mM ¹⁴C-Acetyl CoA. The product is isolated by binding tophosphocellulose filter paper and the extent of reaction is determinedby scintillation counting.

As shown in FIG. 26, DACS inhibited spermidine/spermineacetyltransferase (SSAT) in a dose-related manner.

EXAMPLE XXII Tricyclic and other Heterocyclic Compounds Can InhibitPolyamine Transport

Employing the polyamine transport assay described in Example XX, severalheterocyclic ring compounds were tested for their activity as inhibitorsof transport. The unexpected discovery was made that that severalcompounds strongly resembling tricyclic antidepressants andantipsychotic agents inhibited polyamine transport. Of the compoundsshown in FIG. 25 compounds 161, 162 and 165 inhibited the PAT assay inboth A172 and MDA cell lines. Compound 165 acted as a non-competitiveinhibitor of PAT with a K_(i) of 41 nM (for A172 cells) and 500 nM (forMDA cells).

These compounds resembled compounds 163-164 in FIG. 25 which are knownantipsychotic and antidepressant drugs. These observations indicate thatthat compounds of this type modulate polyamine uptake.

EXAMPLE XXIII Effect of Linker Length or “Headless” Status on GrowthInhibition by Polyamine Analogues

Compounds were tested for their ability to inhibit cell growth in thepresence of 1 μM spermidine and 230 μM ODC inhibitor for the MDA-MB-231cells or 1 mM ODC inhibitor for the PC3 cells. Cells were plated anddrugs were added as described in Example XIX. “Headless” linkers withcarbons of 2 or 3 chain length were ineffective on the MDA-MB-231 breastcarcinoma but inhibited growth in the PC3 prostatic carcinoma cells asshown in FIGS. 19 and 20

EXAMPLE XXIV Evaluation of MDS as a Fluorescent Probe in a PAT Assay

The goal of this experiment was to show that MDS competes with³H-spermidine in a transport assay.

Using the general radiometric PAT assay and A172 cells as describedabove, MDS was found to competitively displace ³H-spermidine in thetransport assay (FIGS. 27 and 28).

EXAMPLE XXV Fluorescent Microscopic Analysis of MonodansylsperrnineUptake

Cells were plated in a sterile chambered slide and grown for 15-48 hoursto assure adherence of cells to the slide. The medium was removed andreplaced with fresh medium containing 1 μM MDS for a 10 minuteincubation period at 37° C. The medium was then removed and the cellswashed 3 times with phosphate buffered saline. Glycerol (50% v/v) in avolume of 50 μl was added to the chamber, and the slide was removed andcovered with a cover slip.

Using a fluorescence microscope with filters set for excitation at 340nm and emission at 530 nm, the slide was observed under normal light andwith fluorescence. Uptake of the dansylated spermine was observedmicroscopically and recorded on photograph.

Although a photomicrograph is not included here, cultured cellsincubated with MDS took up the labeled material as indicated by thefluorescence which was visualized microscopically. Nucleoli, whichcontain large amounts of RNA to which the probe could bind, showedparticularly strong staining. As expected, the probe was seen lining themembranous structures.

EXAMPLE XXVI Enzymatic Detection of N¹-dansylspermine

Polylysine plates were prepared by addition of 200 μl of polylysine (5μg/ml) in 10 mM Tris-HCl buffer, pH 8.5, containing 10 mM NaCl and 10 mMNaN₃ The plates were incubated for 20 min at 37° C. when the wells werewashed 3× with 200 pi water. The plates were then treated with μ l of2.5% glutaraldehyde in 50 mM borate buffer pH 10.0 for 1 hr at 25° C.,when the wells were washed with 200 μl of 50 mM borate buffer pH 10.0twice and once with water. Various concentrations of eitherN1-dansylspermine or DACS were added to the wells ranging between 0.1and 10 pmoles/well and incubated for 1 hr at room temperature. Theplates were then washed with twice with 200 μl or PBS. The wells werethen treated with 200 μl of a 0.3% NH₄OH in PBS and was incubated for 1hr at room temperature when it was washed twice with 200 μl of PBS-0.5%Tween (PBST). The wells were then treated with 200 μl of 0.5% NABH₄ inPBS for 10 minutes when they were washed twice with 200 μl PBST. Thewells were then blocked with 200 μl 1% BSA for 1 hour when they werewashed once with PBST. Dansyl anti-body (Molecular Probes, Eugene,Oreg.) was added at a {fraction (1/200)} dilution to each well in 100 μlPBST and incubated overnight at 4° C. when it was washed four times withPBST. To each well was now added 100 μl of anti-HRP antibody at a{fraction (1/5000)} dilution and incubated for 2 hours at 4° C. wheneach well was washed four times with PBST. Enzyme activity wasdetermined using either 100 μl of NBT or OPD (5 mg OPD/10 ml of 0.1Mcitrate buffer, pH 5.0) and an incubation period of 10 minutes at roomtemperature. The color was measured at 630 nm in a plate reader.

This method is an alternate embodiment of the of the PAT assay usingindirect detection to enhance the signal and lower the detection limits.This method allows for the detection of extremely low concentrations ofprobe. The results, shown in FIG. 32, indicated that DACS levels as lowas 0.1 pmoles could be detected.

EXAMPLE XXVII Modifications of Polyamine Analogues

By “modifying” the extending polyamine analogues with aldehydicnucleoside terminators, it is possible to produce sequence specifichybrid oligomers. Each amino group is “modified” individually andspecifically with any of the four ribonucleosides (or2′-deoxyribonucleosides) as shown in FIG. 38.

This technology provides an approach for solving the problem oftriple-helix forming antisense oligonucleotides (Chan, P. P. et al., J.Mol. Med. 75: 267-282 (1997) by combining the transportability ofpolyamines into cells with structural features of nucleotide sequencespecificity. The transport overcomes the limitations of bioavailabilitywhile also enhancing the bio-stability of such an oligomer.

EXAMPLE XXVIII

Using the approach outlined in FIGS. 36 and 37, compound 31a (FIG. 39)is synthesized using the blocked 3-aminopropanal 27a, benzaldehyde 28aas the first terminator, the blocked methioninal 29a as an extender andacetone as the final terminator.

EXAMPLE XXIX

A library of compounds is synthesized by using the appropriate blockedaminoaldehydes, aldehydes or ketones. The general structures are shownbelow.

In the case of aldehydes and aminoaldehydes, R1 and R3 are bothhydrogen. In the case of ketones and aminoketones R₁=R₃=H or—(CH₂)_(n)CH₃ where n=0 to 6. The keto-function can also be a part of aring structure. R₂ and R₄ can be aliphatic, alicyclic, aromatic andheterocyclic. Examples of compounds that could be contain aldehyde,ketone, amino-aldehyde or amino-ketone functions are dibenzofuran,acridine, 2,1,3-benzothiodiazole, quinoline, isoquinoline, benzofuran,indole, carbazole, fluorene, 1,3-benzodiazine, phenazine, phenoxazine,phenothiazine, adamantane, camphor, piperidine, alkylpiperazine,morpholine, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, thiophene, furan, pyrrole, alkyl-1,2-diazole,alkylimidazole, alkyl-1H-1,2,3-triazol, alkyl-1H1,2,3,4-tetrazole,thiazole, oxazole, 1,3,4-thiadiazole, pyridinyl, pyrimidine,1,2-diazine, 1,4-diazine and 1,3,5-triazine, 4-dimethylaminoazobenzene,2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole, benzene, naphthalene,phenanthrene, anthracene, pyrene, alkanes containing 2 to 10 carbons,alkenes containing 1 to 3 unsaturations and 3 to 10 carbons, alkynescontaining 1 to 3 unsaturations and 3 to 10 carbons, branched alkanes,alkenes, alkynes containing 3 to 10 carbon atoms. Many aldehydes,ketones, aminoaldehydes and aminoketones containing one or more of thefunctional groups listed above, are commercially available. A number ofaminoalcohols, precursors for aminoaldehydes are listed in Table 2,below.

TABLE 2 Aminoalcohol Extenders Alinol 2-amino-2-methyl-1-propanolL-methioninol D-glucosamine R,S-2-amino-1-butanol 4-aminobutanol3-amino-1-propanol trans--2-aminocy clohexanol 5-aminopentanol(S)-(+)-2-amino-3-cyclohexyl-1- propanol R,S-2-amino-2-phenylethanolDL-2-amino-1-hexanol 6-amino-1-hexanol 1-(1S,2S)(+)2-amino-3-methoxy-1-phenyl-1-propanol 2-amino-3-methyl-1-pentanol2-amino-4-methyl-1-pentanol 2-(2-amino-4-nitroanilino)ethanolD,L-2-amino-1-pentanol 2-aminophenethyl alcohol2-amino-1-phenethylethanol 2-amino-3-methyl-1-pentanol(R)-(+)-2-amino-3- phenyl--1-propanol (S)-(−)-2-amino-3-2-(-3aminophenylsulfonyl)ethanol phenyl--1-propanolD,L-1-amino-2-propanol D,L-2-amino-1-propanol 3-amino-1-propanolD-galactosamine D-mannosamine

EXAMPLE XXX

A library of compounds is synthesized by using the appropriate blockedaminoaldehydes, aldehydes or ketones selected from commerciallyavailable sources or from synthetic routes known in the art.Aminoaldehydes are synthesized in a variety of ways from variousstarting materials such as L-and D-amino acids, aminoalcohols, oralcohols or carboxylic acid substituted with NO₂ or —CN groups.Aminoaldehydes are synthesized from appropriately blocked aminoalcoholsby known procedures (Larack, R., In: Comprehensive OrganicTransformations, VCH Publishers, Inc., NY, 1989, pp. 604-616).Aminoaldehydes are directly synthesized from appropriately blockedaminocarboxylic acids or blocked aminonitrile (supra at p. 616-617).

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth as follows in the scope ofthe appended claims.

What is claimed is:
 1. A method for the synthesis of a polyamineanalogue by chain extension comprising 1) attaching a cleavable linkerto activate a soluble or insoluble support followed sequentially byattaching, to said linker, one or more extender synthons comprising freeamino and protected aldehyde moieties, and attaching, to the lastextender synthon, a chain terminator so that a desired polyaminecontaining chain is present; and 2) cleaving the polyamine chain fromthe support, wherein said attached synthons may be additionally reducedby reductive amidation.
 2. The method of claim 1 wherein said attachingstep comprises a) attaching to the soluble or insoluble support acleavable linker containing a first amide bond with an activated leavingmoiety; b) attaching via the first amide bond a first extender synthoncontaining a free amino moiety and a protected aldehyde moiety; c)deprotecting the protected aldehyde moiety; d) optionally attaching, viathe deprotected aldehyde moiety, one or more additional extendersynthons by repeating steps b) and c), where step b) is reductiveamination; and e) reacting the deprotected aldehyde moiety with an amineunder reductive amination conditions and attaching an aldehydecontaining chain terminator; wherein reductive amination of a secondaryamine with an aldehyde in the extending chain may optionally occur aftereach attaching step b).
 3. The method of claim 1 or 2 wherein thecleavable linker yields polyamine chains with an alcohol group, amide orsubstituted amides upon cleavage.
 4. The method of claim 1 or 2 whereinthe support is selected from the group consisting of MeO-polyethyleneglycol-OH, 3,4-dihydro-2H-pyran-2-yl-methoxymethyl polystyrene,polystyrene resins, chip-based systems, multi-pin systems and hydroxylgroup containing microwells; the extender synthon is a free amino andprotected aldehyde moieties containing form of a compound selected fromthe group consisting of chiral amino acids and amino acid precursors,reactive moieties that bind polyamine binding molecules, aliphaticstructures, aromatic structures, heterocyclic structures, carbohydrates,nucleosides, and known drug agents, each of which contains a free aminomoiety and a protected aldehyde moiety; and the chain terminator isselected from the group consisting of acrolein, unsaturated alkenealdehydes, straight chain alkyl aldehydes, and branched chain alkylaldehydes.
 5. The method of claim 4 wherein the extender synthon is afree amino and protected aldehyde moieties containing form of a compoundselected from the group consisting of Alinol 2-amino-2-methyl-1-propanolL-methioninol D-glucosamine R,S-2-amino-1-butanol 4-aminobutanol3-amino-1-propanol trans--2-aminocy clohexanol 5-aminopentanol(S)-(+)-2-amino-3-cyclohexyl-1- propanol R,S-2-amino-2-phenylethanolDL-2-amino-1-hexanol 6-amino-1-hexanol 1-(1S,2S)(+)2-amino-3-methoxy-1-phenyl-1-propanol 2-amino-3-methyl-1-pentanol2-amino-4-methyl-1-pentanol 2-(2-amino-4-nitroanilino)ethanolD,L-2-amino-1-pentanol 2-aminophenethyl alcohol2-amino-1-phenethylethanol 2-amino-3-methyl-1-pentanol(R)-(+)-2-amino-3- phenyl--1-propanol (S)-(−)-2-amino-3-2-(-3aminophenylsulfonyl)ethanol phenyl--1-propanolD,L-1-amino-2-propanol D,L-2-amino-1-propanol 3-amino-1-propanolD-galactosamine and D-mannosamine.


6. The method of claim 1 or 2 wherein the steps are conducted inparallel using more than one extender synthon and more than one chainterminator.
 7. A library of polyamine derivatives produced by the methodof claim
 6. 8. The library of claim 7, wherein said derivatives have theformula R₁—X—R₂ wherein R₁ is H, or is a head group selected from thegroup consisting of a straight or branched C₁₋₁₀ aliphatic, alicyclic,single or multring aromatic, single or multiring aryl substitutedaliphatic, aliphatic-substituted single or multiring aromatic, a singleor multiring heterocyclic, a single or multiringheterocyclic-substituted aliphatic and an aliphatic-substitutedaromatic; R₂ is a polyamine; and X is CO, NHCO, NHCS, or SO₂.
 9. Alibrary according to claim 8 wherein R₂ has the formulaNH(CH₂)_(n)NH(CH₂)_(p)NH(CH₂)_(q)NHR₃ wherein (a) n, p and q varyindependently and n=p=q=1 to 12; (b) R₃ is H; C₁₋₁₀ alkyl; C₁₋₁₀alkenyl; C₁₋₁₀ alkynyl; alicyclic; aryl; aryl-substituted alkyl, alkenylor alkynyl; alkyl-, alkenyl-, or alkynyl-substituted aryl; gauanidino;heterocyclic; heterocyclic-substituted alkyl, alkenyl or alkynyl; andalkyl-, alkenyl-, or alkynyl-substituted heterocyclic.
 10. A libraryaccording to claim 8, wherein the derivatives further comprise, linkedbetween X and R₂, a linker L and an additional group y, such that thederivatives have the formula: R₁—X—L—Y—R₂ wherein, L is a C₁₋₁₀ alkyl,C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, alicyclic, or heterocyclic; X is CO, SO₂,NHCO or NHCS; and Y is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O,or S.
 11. A library according to claim 8, where R₁ has the formula:

wherein R₄, R₅, R₆, R₇ and R₈ are, independently, H, OH, halogen, NO₂,NH₂, NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH₂)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ wheren=0 to
 10. 12. A library according to claim 8 where R₁ has the formula:

wherein R₄ and R₅ are, independently, H, OH, halogen, NO₂, NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH₂)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃, wheren=0 to
 10. 13. A library according to claim 8 wherein R₁ has theformula:

wherein r and s vary independently and r=s=0 to 6; R₄, R₅, R₆, R₇, R₈and R₉ are, independently, H, OH, halogen, NO₂, NH₂, NH(CH)_(n)CH₃,N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH₂)_(n)CH₃,NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ where n=0 to 10; andQ is CONH, SO₂NH, CO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.
 14. A libraryaccording to claim 8 wherein R₁ has the formula:

wherein r and s vary independently and are 0 to 6; R₄, R₅, R₆ and R₇are, independently, H, OH, NO₂, NH₂, NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN,(CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH₂)_(n)CH₃, NCO(CH₂)_(n)CH₃,O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ where n=0 to 10; and Q is CONH, SO₂NH,NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.
 15. A library according toclaim 8, wherein R₁ is selected from the group consisting ofnaphthalene, phenanthrene, anthracene, pyrene, dibenzofuran, acridine,2,1,3-benzothiodiazole, quinoline, isoquinoline, benzofuran, indole,carbazole, fluorene, 1,3-benzodiazine, phenazine, phenoxazine,phenothiazine, adamantane, camphor, pipiridine, alkylpiperazine,morpholine, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, thiophene, furan, pyrrole, alkyl-1,2-diazole,alkylimidazole, alkyl-1H-1,2,3-triazol, alkyl-1H1,2,3,4-tetrazole,thiazole, oxazole, 1,3,4-thiadiazole, pyridinyl, pyrimidine,1,2-diazine, 1,4-diazine and 1,3,5-triazine, 4-dimethylaminoazobenzene,3-phenyl-5-methylisooxazole, 3-(2-chlorophenyl)-5-methylisooxazole,2-(4-chloropheny)-6-methyl-7-chloroquinoline,6-chloroimidazo[2,1-β]thiazole, α-methylcinnamic acid, and2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole.
 16. A library accordingto claim 8 wherein R₁ is a D- or L-amino acid.
 17. A library accordingto claim 8 where R₁ has the formula selected from the group consistingof (A) R₁₂—R₁₃—Y₁—R₁₄ (B) R₁₂Y₁R₁₃Z₁R₁₄

wherein R₁₂ and R₁₃, independently, are H, naphthalene, phenanthrene,anthracene, pyrene, dibenzofuran, acridine, 2,1,3-benzothiodiazole,quinoline, isoquinoline, benzofuran, indole, carbazole, fluorene,1,3-benzodiazine, phenazine, phenoxazine, phenothiazine, adamantane,camphor, pipiridine, alkylpiperazine, morpholine, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, thiophene,furan, pyrrole, alkyl-1,2-diazole, alkylimidazole,alkyl-1H-1,2,3-triazol, alkyl-1H1,2,3,4-tetrazole, thiazole, oxazole,1,3,4-thiadiazole, pyridinyl, pyrimidine, 1,2-diazine, 1,4-diazine and1,3,5-triazine, 4-dimethylaminoazobenzene, 3-phenyl-5-methylisooxazole,3-(2-chlorophenyl)-5-methylisooxazole,2-(4-chloropheny)-6-methyl-7-chloroquinoline,6-chloroimidazo[2,1-β]thiazole, α-methylcinnamic acid, or2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole; and further, wherein aring of R₁₂, R₁₃ or both in formulas (A), (B) and (D), is optionallysubstituted with one or more of OH, halogen, NO₂, NH₂, NH(CH)_(n)CH₃,N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH₂)_(n)CH₃,NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or COO(CH)_(n)CH₃, where n=0 to 10; R₁₄and R₁₅, and, in formula (C), R₁₃, independently, are (CH₂)_(n),(CH₂)_(n)CH═CH, (CH₂)_(n)(CH═CH)_(m)CO, or (CH₂)_(n)CO where n=0 to 5and m=1 to 3; Y₁ and Z₁, independently, are CONH, SO₂NH, NHCO, NHCONH,NHCSNH, NHSO₂′ NHSO₂, SO₂—NHSO₂, SO₂, O, S, COO or when R₁ is of formula(A) or (B), Y₁ represents a bond between a C or N atom of R₁₂ and a C orN atom of R₁₃ and Z₁ represents a bond between a C or N atom of R₁₃ anda C or N atom of R₁₄; or when R₁ is of formula (C) or Y₁ represents abond between the C and a C or N atom of R₁₃ and Z₁ represents a bondbetween the C and a C or N atom of R₁₄; or when R₁ is of formula (D) Y₁represents a bond between a C or N atom of R₁₂ and a C or N atom of R₁₄and Z₁ represents a bond between a C or N atom of R₁₃ and a C or N atomof R₁₅.
 18. A library according to claim 8 wherein R₂has the formulaNHCH(Z₁)(CH₂)_(n)NH(CH₂)_(p)NH(CH₂)_(q)CH(Z₁)NHR₃ and wherein (a) n, pand q vary independently and n=p=q=1 to 12; (b) R₃ is H; C₁₋₁₀ alkyl;C₁₋₁₀ alkenyl; C₁₋₁₀ alkynyl; alicyclic; aryl; aryl-substituted alkyl,alkenyl or alkynyl; alkyl-, alkenyl-, or alkynyl-substituted aryl;gauanidino or heterocyclic; and (c) Z₁ is CH₃, CH₂CH₃ or cyclopropyl.19. A library according to claim 8 wherein R₂ has the formula:

and wherein x=1 to 4; y=1 to 3, R₁₀ and R₁₁ are, independently, H,(CH₂)_(n)NHR₁₂ or (CH₂)_(k)NH(CH₂)₁ NHR₁₂ where n=k=1=1 to 10, and R₁₂is H or C(N═H)NH₂.
 20. A library according to claim 8 wherein R₂ isselected from the group consisting of N¹-acetylspermine,N¹-acetylspermidine, N⁸-acetylspermidine, N¹-guanidinospermine,cadaverine, aminopropylcadaverine, homospermidine, caldine(horspermidine), 7-hydroxyspermidine, thermine (norspermine),thermospermine, canavalmine, aminopropylhomospermidine,N,N′-bis(3-aminoppropyl)cadaverine, aminopentylnorspermidine,N⁴-aminopropylnorspermidine, N⁴-aminopropylspermidine, caldopentamine,homocaldopentamine, N⁴-bis(aminopropyl)norspermidine, thermopentamine,N⁴-bis(aminopropyl)spermidine, caldohexamine, homothermohexamine,homocaldohexamine, N-(3-aminopropyl)-1,3-propanediamine,N,N′-bis(3-aminopropyl)ethylendiamine,N,N′-bis(3-aminopropyl)-1,4-piperazine,N,N′-bis(3-aminopropyl)-1,3-piperazine,N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-bis(2-aminoethyl)-1,3-propanediamine, tris(3-aminopropyl)amine, andtris(aminoethyl)amine.
 21. A library according to claim 8 wherein saidlibrary comprises one or more polyamine analogues selected from thegroup consisting of compounds designated in FIG. 2 as 3, 4, 5, 6, 13,14, 29, 40, 43, 44, 45, 57, 58, 56, 66, 67, 72, 76, 84, 88, 89, 95 and96.
 22. A library according to claim 21, wherein said library comprisesone or more polyamine analogues selected from the group consisting ofcompounds designated in FIG. 2 as 4, 5, 6, 43, 65, 66, 84, 89, 95 or 96.23. A library of polyamine compounds comprising one or more polyamineanalogues selected from the group consisting of compounds designated inFIG. 2 as 3, 4, 5, 6, 13, 14, 29, 40, 43, 44, 45, 57, 58, 56, 66, 67,72, 76, 84, 88, 89, 95 and
 96. 24. The library according to claim 23,wherein said library comprises one or more polyamine analogues selectedfrom the group consisting of compounds designated in FIG. 2 as 4, 5, 6,43, 65, 66, 84, 89, 95 or 96.